Compositions and methods for reverse transcription of nucleic acid molecules

ABSTRACT

The present invention is generally related to compositions and methods for the reverse transcription of nucleic acid molecules, especially messenger RNA molecules. Specifically, the invention relates to compositions comprising mixtures of polypeptides having reverse transcriptase (RT) activity, and to methods of producing, amplifying or sequencing nucleic acid molecules (particularly cDNA molecules) using these compositions or polypeptides, particularly at temperatures above about 55° C. The invention also relates to nucleic acid molecules produced by these methods, to vectors and host cells comprising these nucleic acid molecules, and to the use of such nucleic acid molecules to produce desired polypeptides. The invention also relates to methods for producing Rous Sarcoma Virus (RSV) and Avian Myeloblastosis Virus (AMV) RTs or other Avian Sarcoma-Leukosis Virus (ASLV) RTs (α and/or β subunits thereof), to isolated nucleic acid molecules encoding such RSV RT, AMV RT or other ASLV RT subunits, to vectors and host cells comprising these isolated nucleic acid molecules and to RSV RT, AMV RT and other ASLV RT subunits produced by these methods. The invention further relates to nucleic acid molecules encoding recombinant heterodimeric RT holoenzymes, particularly heterodimeric RSV RTs, AMV RTs or other ASLV RTs (which may be αβ RTs, ββ RTs, or α RTs), vectors (particularly baculovirus vectors) and host cells (particularly insect and yeast cells) comprising these nucleic acid molecules, methods for producing these heterodimeric RTs and heterodimeric RTs produced by these methods. The invention also relates to kits comprising the compositions, polypeptides, or RSV RTs, AMV RTs or other ASLV RTs of the invention.

CROSS REFERENCE TO RELATED APPLICATIONS

[0001] The present application claims the benefit of U.S. ProvisionalApplication No. 60/044,589, filed Apr. 22, 1997, and No. 60/049,874,filed Jun. 17, 1997, the disclosures of which are entirely incorporatedby reference herein.

FIELD OF THE INVENTION

[0002] The present invention is in the fields of molecular and cellularbiology. The invention is generally related to reverse transcriptaseenzymes and methods for the reverse transcription of nucleic acidmolecules, especially messenger RNA molecules. Specifically, theinvention relates to compositions comprising mixtures of reversetranscriptase enzymes, and to methods of producing, amplifying orsequencing nucleic acid molecules (particularly cDNA molecules) usingthese reverse transcriptase enzymes or compositions. The invention alsorelates to nucleic acid molecules produced by these methods and to theuse of such nucleic acid molecules to produce desired polypeptides. Theinvention also concerns kits comprising such compositions.

BACKGROUND OF THE INVENTION

[0003] cDNA and cDNA Libraries

[0004] In examining the structure and physiology of an organism, tissueor cell, it is often desirable to determine its genetic content. Thegenetic framework of an organism is encoded in the double-strandedsequence of nucleotide bases in the deoxyribonucleic acid (DNA) which iscontained in the somatic and germ cells of the organism. The geneticcontent of a particular segment of DNA, or gene, is only manifested uponproduction of the protein which the gene encodes. In order to produce aprotein, a complementary copy of one strand of the DNA double helix (the“coding” strand) is produced by polymerase enzymes, resulting in aspecific sequence of ribonucleic acid (RNA). This particular type ofRNA, since it contains the genetic message from the DNA for productionof a protein, is called messenger RNA (mRNA).

[0005] Within a given cell, tissue or organism, there exist myriad mRNAspecies, each encoding a separate and specific protein. This factprovides a powerful tool to investigators interested in studying geneticexpression in a tissue or cell—mRNA molecules may be isolated andfurther manipulated by various molecular biological techniques, therebyallowing the elucidation of the full functional genetic content of acell, tissue or organism.

[0006] One common approach to the study of gene expression is theproduction of complementary DNA (cDNA) clones. In this technique, themRNA molecules from an organism are isolated from an extract of thecells or tissues of the organism. This isolation often employs solidchromatography matrices, such as cellulose or agarose, to whicholigomers of thymidine (T) have been complexed. Since the 3′ termini onmost eukaryotic mRNA molecules contain a string of adenosine (A) bases,and since A binds to T, the mRNA molecules can be rapidly purified fromother molecules and substances in the tissue or cell extract. From thesepurified mRNA molecules, cDNA copies may be made using the enzymereverse transcriptase (RT), which results in the production ofsingle-stranded cDNA molecules. The single-stranded cDNAs may then beconverted into a complete double-stranded DNA copy (i.e., adouble-stranded cDNA) of the original mRNA (and thus of the originaldouble-stranded DNA sequence, encoding this mRNA, contained in thegenome of the organism) by the action of a DNA polymerase. Theprotein-specific double-stranded cDNAs can then be inserted into aplasmid or viral vector, which is then introduced into a host bacterial,yeast, animal or plant cell. The host cells are then grown in culturemedia, resulting in a population of host cells containing (or in manycases, expressing) the gene of interest.

[0007] This entire process, from isolation of mRNA to insertion of thecDNA into a plasmid or vector to growth of host cell populationscontaining the isolated gene, is termed “cDNA cloning.” If cDNAs areprepared from a number of different mRNAs, the resulting set of cDNAs iscalled a “cDNA library,” an appropriate term since the set of cDNAsrepresents a “population” of genes comprising the functional geneticinformation present in the source cell, tissue or organism. Genotypicanalysis of these cDNA libraries can yield much information on thestructure and function of the organisms from which they were derived.

[0008] Retroviral Reverse Transcriptase Enzymes

[0009] Three prototypical forms of retroviral RT have been studiedthoroughly. Moloney Murine Leukemia Virus (M-MLV) RT contains a singlesubunit of 78 kDa with RNA-dependent DNA polymerase and RNase Hactivity. This enzyme has been cloned and expressed in a fully activeform in E. coli (reviewed in Prasad, V. R., Reverse Transcriptase, ColdSpring Harbor, N.Y.: Cold Spring Harbor Laboratory Press, p.135 (1993)).Human Immunodeficiency Virus (HIV) RT is a heterodimer of p66 and p51subunits in which the smaller subunit is derived from the larger byproteolytic cleavage. The p66 subunit has both a RNA-dependent DNApolymerase and an RNase H domain, while the p51 subunit has only a DNApolymerase domain. Active HIV p66/p51 RT has been cloned and expressedsuccessfully in a number of expression hosts, including E. coli(reviewed in Le Grice, S. F. J., Reverse Transcriptase, Cold SpringHarbor, N.Y.: Cold Spring Harbor Laboratory press, p. 163 (1993)).Within the HIV p66/p51 heterodimer, the 51-CKD subunit is catalyticallyinactive, and the 66-kD subunit has both DNA polymerase and RNase Hactivity (Le Grice, S. F. J., et al., EMBO Journal 10:3905 (1991);Hostomsky, Z., et al., J. Virol. 66:3179 (1992)). Avian Sarcoma-LeukosisVirus (ASLV) RT, which includes but is not limited to Rous Sarcoma Virus(RSV) RT, Avian Myeloblastosis Virus (AMV) RT, Avian ErythroblastosisVirus (AEV) Helper Virus MCAV RT, Avian Myelocytomatosis Virus MC29Helper Virus MCAV RT, Avian Reticuloendotheliosis Virus (REV-T) HelperVirus REV-A RT, Avian Sarcoma Virus UR2 Helper Virus UR2AV RT, AvianSarcoma Virus Y73 Helper Virus YAV RT, Rous Associated Virus (RAV) RT,and Myeloblastosis Associated Virus (MAV) RT, is also a heterodimer oftwo subunits, a (approximately 62 kDa) and β (approximately 94 kDa), inwhich a is derived from P by proteolytic cleavage (reviewed in Prasad,V. R., Reverse Transcriptase, Cold Spring Harbor, N.Y.: Cold SpringHarbor Laboratory Press (1993), p. 135). ASLV RT can exist in twoadditional catalytically active structural forms, ββ and α (Hizi, A. andJoklik, W. K., J. Biol. Chem. 252: 2281 (1977)). Sedimentation analysissuggests αβ and ββ are dimers and that the a form exists in anequilibrium between monomeric and dimeric forms (Grandgenett, D. P., etal., Proc. Nat. Acad. Sci. USA 70: 230 (1973); Hizi, A. and Joklik, W.K., J. Biol. Chem. 252: 2281 (1977); and Soltis, D. A. and Skalka, A.M., Proc. Nat. Acad. Sci. USA 85: 3372 (1988)). The ASLV αβ and ββ RTsare the only known examples of retroviral RT that include threedifferent activities in the same protein complex: DNA polymerase, RNaseH, and DNA endonuclease (integrase) activities (reviewed in Skalka, A.M., Reverse Transcriptase, Cold Spring Harbor, N.Y.: Cold Spring HarborLaboratory Press (1993), p. 193). The α form lacks the integrase domainand activity.

[0010] Various forms of the individual subunits of ASLV RT have beencloned and expressed. These include a 98-kDa precursor polypeptide thatis normally processed proteolytically to β and a 4-kDa polypeptideremoved from the p carboxy end (Alexander, F., et_al., J. Virol. 61: 534(1987) and Anderson, D. et al., Focus 17:53 (1995)), and the mature βsubunit (Weis, J. H. and Salstrom, J. S., U.S. Pat. No. 4, 663, 290(1987); and Soltis, D. A. and Skalka, A. M., Proc. Nat. Acad. Sci. USA85:3372 (1988)). Heterodimeric RSV αβ RT has also been purified from E.coli cells expressing a cloned RSV β gene (Chernov, A. P., et al.,Biomed. Sci. 2:49 (1991)). However, there have been no reportsheretofore of the simultaneous expression of cloned ASLV RT α and βgenes resulting in the formation of heterodimeric αβ RT.

[0011] Reverse Transcription Efficiency

[0012] As noted above, the conversion of mRNA into cDNA by RT-mediatedreverse transcription is an essential step in the study of proteinsexpressed from cloned genes. However, the use of unmodified RT tocatalyze reverse transcription is inefficient for at least two reasons.First, RT sometimes destroys an RNA template before reversetranscription is initiated, primarily due to the activity of intrinsicRNase H activity present in RT. Second, RT often fails to completereverse transcription after the process has been initiated (Berger, S.L., et al., Biochemistry 22:2365-2372 (1983); Krug, M. S., and Berger,S. L., Meth. Enzymol. 152:316 (1987)). Removal of the RNase H activityof RT can eliminate the first problem and improve the efficiency ofreverse transcription (Gerard, G. F., et al., FOCUS 11(4):60 (1989);Gerard, G. F., et al., FOCUS 14(3):91 (1992)). However RTs, includingthose forms lacking RNase H activity (“RNase H⁻” forms), still tend toterminate DNA synthesis prematurely at certain secondary structural(Gerard, G. F., et al., FOCUS 11(4):60 (1989); Myers, T. W., andGelfand, D. H., Biochemistry 30:7661 (1991)) and sequence (Messer, L.I., et al., Virol. 146:146 (1985)); Abbotts, J., et al., J. Biol. Chem.268:10312-10323 (1993)) barriers in nucleic acid templates.

[0013] Even in the most efficient reverse transcription systemsavailable today, which use RNase H⁻ M-MLV RT, yields of total cDNAproduct generally do not exceed 50% of input mRNA and the fraction ofthe product that is full-length does not exceed 50%. The secondarystructural and sequence barriers in the mRNA template, which asdescribed above can give rise to these limitations, occur frequently athomopolymer stretches (Messer, L. I., et al., Virol. 146:146 (1985);Huber, H. E., et al., J. Biol. Chem. 264:4669-4678 (1989); Myers, T. W.,and Gelfand, D. H., Biochemistry 30:7661 (1991)), are more oftensequence rather than secondary structural barriers (Abbotts, J., et al.,J. Biol. Chem. 268:10312-10323 (1993)), and are often distinct fordifferent RTs (Abbotts, J., et al., J. Biol. Chem. 268:10312-10323(1993)). If these barriers could be overcome, yield of total andfull-length cDNA product in reverse transcription reactions could beincreased.

SUMMARY OF THE INVENTION

[0014] The present invention provides reverse transcriptase enzymes,compositions comprising such enzymes and methods useful in overcomingthe above-described cDNA length limitations. In general, the inventionprovides compositions for use in reverse transcription of a nucleic acidmolecule comprising two or more different polypeptides having reversetranscriptase activity. Such compositions may further comprise one ormore nucleotides, a suitable buffer, and/or one or more DNA polymerases.The compositions of the invention may also comprise one or moreoligonucleotide primers. Each reverse transcriptase used in thecompositions of the invention may have a different transcription pausesite on a given mRNA molecule. The reverse transcriptases in thesecompositions preferably are reduced or substantially reduced in RNase Hactivity, and most preferably are enzymes selected from the groupconsisting of Moloney Murine Leukemia Virus (M-MLV) H⁻ reversetranscriptase, Rous Sarcoma Virus (RSV) H⁻ reverse transcriptase, AvianMyeloblastosis Virus (AMV) H⁻ reverse transcriptase, Rous AssociatedVirus (RAV) H⁻ reverse transcriptase, Myeloblastosis Associated Virus(MAV) H⁻ reverse transcriptase and Human Immunodeficiency Virus (HIV) H⁻reverse transcriptase or other ASLV H⁻ reverse transcriptases. Inpreferred compositions, the reverse transcriptases are present atworking concentrations.

[0015] The invention is also directed to methods for making one or morenucleic acid molecules, comprising mixing one or more nucleic acidtemplates (preferably one or more RNA templates and most preferably oneor more messenger RNA templates) with two or more polypeptides havingreverse transcriptase activity and incubating the mixture underconditions sufficient to make a first nucleic acid molecule or moleculescomplementary to all or a portion of the one or more nucleic acidtemplates. In a preferred embodiment, the first nucleic acid molecule isa single-stranded cDNA. Nucleic acid templates suitable for reversetranscription according to this aspect of the invention include anynucleic acid molecule or population of nucleic acid molecules(preferably RNA and most preferably mRNA), particularly those derivedfrom a cell or tissue. In a preferred aspect, a population of mRNAmolecules (a number of different mRNA molecules, typically obtained fromcells or tissue) are used to make a cDNA library, in accordance with theinvention. Preferred cellular sources of nucleic acid templates includebacterial cells, fungal cells, plant cells and animal cells.

[0016] The invention also concerns methods for making one or moredouble-stranded nucleic acid molecules. Such methods comprise (a) mixingone or more nucleic acid templates (preferably RNA or mRNA, and morepreferably a population of mRNA templates) with two or more polypeptideshaving reverse transcriptase activity; (b) incubating the mixture underconditions sufficient to make a first nucleic acid molecule or moleculescomplementary to all or a portion of the one or more templates; and (c)incubating the first nucleic acid molecule under conditions sufficientto make a second nucleic acid molecule or molecules complementary to allor a portion of the first nucleic acid molecule or molecules, therebyforming one or more double-stranded nucleic acid molecules comprisingthe first and second nucleic acid molecules. Such methods may includethe use of one or more DNA polymerases as part of the process of makingthe one or more double-stranded nucleic acid molecules. The inventionalso concerns compositions useful for making such double-strandednucleic acid molecules. Such compositions comprise two or more reversetranscriptases and optionally one or more DNA polymerases, a suitablebuffer and one or more nucleotides.

[0017] The invention also relates to methods for amplifying a nucleicacid molecule. Such amplification methods comprise mixing thedouble-stranded nucleic acid molecule or molecules produced as describedabove with one or more DNA polymerases and incubating the mixture underconditions sufficient to amplify the double-stranded nucleic acidmolecule. In a first preferred embodiment, the invention concerns amethod for amplifying a nucleic acid molecule, the method comprising (a)mixing one or more nucleic acid templates (preferably one or more RNA ormRNA templates and more preferably a population of mRNA templates) withtwo or more different polypeptides having reverse transcriptase activityand with one or more DNA polymerases and (b) incubating the mixtureunder conditions sufficient to amplify nucleic acid moleculescomplementary to all or a portion of the one or more templates.Preferably, the reverse transcriptases are reduced or substantiallyreduced in RNase H activity and the DNA polymerases comprise a first DNApolymerase having 3′exonuclease activity and a second DNA polymerasehaving substantially reduced 3′ exonuclease activity. The invention alsoconcerns compositions comprising two or more reverse transcriptases andone or more DNA polymerases for use in amplification reactions. Suchcompositions may further comprise one or more nucleotides and a buffersuitable for amplification. The compositions of the invention may alsocomprise one or more oligonucleotide primers.

[0018] In accordance with the invention, at least two, at least three,at least four, at least five, at least six, or more, reversetranscriptases may be used. Preferably, two to six, two to five, two tofour, two to three, and most preferably two, reverse transcriptases areused in the compositions and methods of the invention. Such multiplereverse transcriptases may be added simultaneously or sequentially inany order to the compositions or in the methods of the invention.Alternatively, multiple different reactions with different enzymes maybe performed separately and the reaction products may be mixed. Thus,the invention relates to the synthesis of the nucleic acid molecules bythe methods of the invention in which multiple reverse transcriptasesare used simultaneously or sequentially or separately. In particular,the invention relates to a method of making one or more nucleic acidmolecules comprising incubating one or more nucleic acid templates(preferably one or more RNA templates or mRNA templates, and morepreferably a population of mRNA templates) with a first reversetranscriptase under conditions sufficient to make one or more nucleicacid molecules complementary to all or a portion of the one or moretemplates. In accordance with the invention, the one or more nucleicacid molecules (including mRNA templates and/or synthesized nucleic acidmolecules) may be incubated with a second reverse transcriptase underconditions sufficient to make additional nucleic acid moleculescomplementary to all or a portion of the templates or to increase thelength of the previously made nucleic acid molecules. In accordance withthe invention, this procedure may be repeated any number of times withthe same or different reverse transcriptases of the invention. Forexample, the first and second reverse transcriptases may be the same ordifferent. Furthermore, the first and third reverse transcriptases (inaspects of the invention where the procedure is repeated three timesusing a first, second, and third reverse transcriptase) may be the samewhile the second reverse transcriptase may be different from the firstand the third reverse transcriptase. Thus, any combination of the sameand/or different reverse transcriptases may be used in this aspect ofthe invention. Preferably, when multiple reverse transcriptases areused, at least two reverse transcriptases are different.

[0019] In a related aspect of the invention, the reverse transcriptaseused in the reaction may retain all or a portion of its activity duringsubsequent reaction steps. Alternatively, the reverse transcriptase usedin the reaction may be inactivated by any method prior to incubationwith additional reverse transcriptases. Such an inactivation may includebut is not limited to heat inactivation, organic extraction (e.g., withphenol and/or chloroform), ethanol precipitation and the like.

[0020] The synthesized nucleic acid molecules made by simultaneous orsequential or separate addition of reverse transcriptases may then beused to make double stranded nucleic acid molecules. Such synthesizednucleic acid molecules serve as a template which when incubated underappropriate conditions (e.g., preferably in the presence of one or moreDNA polymerases) make nucleic acid molecules complementary to all or aportion of the synthesized nucleic acid molecules, thereby forming anumber of double stranded nucleic acid molecules. The double strandedmolecules may then be amplified in accordance with the invention.

[0021] The invention is also directed to nucleic acid molecules(particularly single- or double-stranded cDNA molecules) or amplifiednucleic acid molecules produced according to the above-described methodsand to vectors (particularly expression vectors) comprising thesenucleic acid molecules or amplified nucleic acid molecules.

[0022] The invention is also directed to recombinant host cellscomprising the above-described nucleic acid molecules, amplified nucleicacid molecules or vectors. Preferred such host cells include bacterialcells, yeast cells, plant cells and animal cells (including insect cellsand mammalian cells).

[0023] The invention is further directed to methods of producing apolypeptide comprising culturing the above-described recombinant hostcells and isolating the polypeptide, and to a polypeptide produced bysuch methods.

[0024] The invention also concerns methods for sequencing one or morenucleic acid molecules using the compositions or enzymes of theinvention. Such methods comprise (a) mixing one or more nucleic acidmolecules (e.g., one or more RNA or DNA molecules) to be sequenced withone or more primers, one or more polypeptides having reversetranscriptase activity, one or more nucleotides and one or moreterminating agents, such as one or more dideoxynucleoside triphosphates;(b) incubating the mixture under conditions sufficient to synthesize apopulation of nucleic acid molecules complementary to all or a portionof the one or more nucleic acid molecules to be sequenced; and (c)separating the population of nucleic acid molecules to determine thenucleotide sequence of all or a portion of the one or more nucleic acidmolecules to be sequenced. In these sequencing methods of the invention,the one or more polypeptides having reverse transcriptase activity maybe added simultaneously, sequentially, or separately to the reactionmixtures as described above.

[0025] The invention is also directed to kits for use in the methods ofthe invention. Such kits can be used for making, sequencing oramplifying nucleic acid molecules (single- or double-stranded). The kitsof the invention comprise a carrier, such as a box or carton, having inclose confinement therein one or more containers, such as vials, tubes,bottles and the like. In the kits of the invention, a first containercontains one or more of the reverse transcriptase enzymes (preferablyone or more such enzymes that are reduced or substantially reduced inRNase H activity) or one or more of the compositions of the invention.In another aspect, the kit may contain one or more containers comprisingtwo or more, three or more, four or more, five or more, six or more, andthe like, reverse transcriptases, preferably one or more containerscomprising two to six, two to five, two to four, two to three, or morepreferably two, reverse transcriptases. The kits of the invention mayalso comprise, in the same or different containers, one or more DNApolymerase (preferably thermostable DNA polymerases), a suitable bufferfor nucleic acid synthesis and one or more nucleotides. Alternatively,the components of the composition may be divided into separatecontainers (e.g., one container for each enzyme). In preferred kits ofthe invention, the reverse transcriptases are reduced or substantiallyreduced in RNase H activity, and are most preferably selected from thegroup consisting of M-MLV H⁻ reverse transcriptase, RSV H⁻ reversetranscriptase, AMV H⁻ reverse transcriptase, RAV H⁻ reversetranscriptase, MAV H⁻ reverse transcriptase and HIV H⁻ reversetranscriptase. In additional preferred kits of the invention, theenzymes (reverse transcriptases and/or DNA polymerases) in thecontainers are present at working concentrations.

[0026] The invention also relates to methods of producing RSV reversetranscriptase (and/or subunits thereof). In particular, the inventionrelates to methods for producing RSV reverse transcriptase (and/orsubunits thereof) containing RNase H activity, to methods for producingRSV reverse transcriptase (and/or subunits thereof) that is reduced orsubstantially reduced in RNase H activity, and to RSV reversetranscriptases (and/or subunits thereof) produced by such methods.

[0027] The invention further relates to methods for using such reversetranscriptases and to kits comprising such reverse transcriptases. Inparticular, the RSV reverse transcriptases (and/or subunits thereof) ofthe invention may be used in methods of sequencing, amplification andproduction (via, e.g., reverse transcription) of nucleic acid molecules.

[0028] The invention also relates to methods of producing AMV reversetranscriptase (and/or subunits thereof). In particular, the inventionrelates to methods for producing AMV reverse transcriptase (and/orsubunits thereof) containing RNase H activity, to methods for producingAMV reverse transcriptase (and/or subunits thereof) that is reduced orsubstantially reduced in RNase H activity, and to AMV reversetranscriptases (and/or subunits thereof) produced by such methods.

[0029] The invention further relates to methods for using such reversetranscriptases and to kits comprising such reverse transcriptases. Inparticular, the AMV reverse transcriptases (and/or subunits thereof) ofthe invention may be used in methods of sequencing, amplification andproduction (via, e.g., reverse transcription) of nucleic acid molecules.

[0030] The invention also generally relates to methods of producing ASLVreverse transcriptases (and/or subunits thereof). In particular, theinvention relates to methods for producing ASLV reverse transcriptases(and/or subunits thereof) containing RNase H activity, to methods forproducing such ASLV reverse transcriptases that are reduced orsubstantially reduced in RNase H activity, and to ASLV reversetranscriptases produced by such methods.

[0031] The invention further relates to methods for using such reversetranscriptases and to kits comprising such reverse transcriptases. Inparticular, the ASLV reverse transcriptases (and/or subunits thereof) ofthe invention may be used in methods of sequencing, amplification andproduction (e.g., via reverse transcription) of nucleic acid molecules.

[0032] The invention further relates to methods for elevated- orhigh-temperature reverse transcription of a nucleic acid moleculecomprising (a) mixing one or more nucleic acid templates (preferably oneor more RNA molecules (e.g., one or more mRNA molecules or polyA+ RNAmolecules, and more preferably a population of mRNA molecules) or one ormore DNA molecules) with one or more polypeptides having reversetranscriptase activity; and (b) incubating the mixture at a temperatureof 50° C. or greater and under conditions sufficient to make a firstnucleic acid molecule or molecules (such as a full length cDNA molecule)complementary to all or a portion of the one or more nucleic acidtemplates. In a preferred aspect, a population of mRNA molecules is usedto make a cDNA library at elevated or high temperatures. In anotheraspect, elevated- or high-temperature nucleic acid synthesis isconducted with multiple reverse transcriptases (i.e., two or more, threeor more, four or more, five or more, six or more, and the like, morepreferably two to six, two to five, two to four, two to three, and stillmore preferably two, reverse transcriptases), which may be added to thereaction mixture simultaneously or sequentially or separately asdescribed above. In preferred such methods, the mixture is incubated ata temperature of about 51° C. or greater, about 52° C. or greater, about53° C. or greater, about 54° C. or greater, about 55° C. or greater,about 56° C. or greater, about 57° C. or greater, about 58° C. orgreater, about 59° C. or greater, about 60° C. or greater, about 61° C.or greater, about 62° C. or greater, about 63° C. or greater, about 64°C. or greater, about 65° C. or greater, about 66° C. or greater, about67° C. or greater, about 68° C. or greater, about 69° C. or greater,about 70° C. or greater, about 71° C. or greater, about 72° C. orgreater, about 73° C. or greater, about 74° C. or greater, about 75° C.or greater, about 76° C. or greater, about 77° C. or greater, or about78° C. or greater; or at a temperature range of from about 50° C. toabout 75° C., about 51° C. to about 75° C., about 52° C. to about 75°C., about 53° C. to about 75° C., about 54° C. to about 75° C., about55° C. to about 75° C., about 50° C. to about 70° C., about 51° C. toabout 70° C., about 52° C. to about 70° C., about 53° C. to about 70°C., about 54° C. to about 70° C., about 55° C. to about 70° C., about55° C. to about 65° C., about 56° C. to about 65° C., about 56° C. toabout 64° C. or about 56° C. to about 62° C. The invention is alsodirected to such methods which further comprise incubating the firstnucleic acid molecule or molecules under conditions sufficient to make asecond nucleic acid molecule or molecules complementary to all orportion of the first nucleic acid molecule or molecules. According tothe invention, the first and second nucleic acid molecules produced bythese methods may be DNA molecules, and may form a double stranded DNAmolecule or molecules which may be a full length cDNA molecule ormolecules, such as a cDNA library. The one or more polypeptides havingreverse transcriptase activity that are used in these methods preferablyare reduced or substantially reduced in RNase H activity, and arepreferably selected from ASLV reverse transcriptases (and/or subunitsthereof) such as one or more subunits of AMV reverse transcriptaseand/or one or more subunits of RSV reverse transcriptase and/or one ormore subunits of MAV reverse transcriptase, and/or one or more subunitsof RAV reverse transcriptase, particularly wherein the subunits arereduced or substantially reduced in RNase H activity.

[0033] The invention also relates to kits for elevated- orhigh-temperature nucleic acid synthesis, which may comprise one or morecomponents selected from the group consisting of one or more reversetranscriptases (preferably one or more ASLV reverse transcriptases suchas AMV or RSV reverse transcriptases (or one or more subunits thereof),and more preferably one or more AMV or RSV reverse transcriptases (orone or more subunits thereof) which are reduced or substantially reducedin RNase H activity), one or more nucleotides, one or more primers andone or more suitable buffers.

[0034] The invention is also directed to nucleic acid molecules producedby the above-described methods which may be full-length cDNA molecules,to vectors (particularly expression vectors) comprising these nucleicacid molecules and to host cells comprising these vectors and nucleicacid molecules.

[0035] Other preferred embodiments of the present invention will beapparent to one of ordinary skill in light of the following drawings anddescription of the invention, and of the claims.

BRIEF DESCRIPTION OF THE DRAWINGS

[0036] FIGS. 1-6 describe, in schematic form (with details omitted forclarity), the construction of the expression vector (pDABH³¹ His) whichplaces the RSV RT a and p genes under control of insect viral promoters:

[0037]FIG. 1: pJD10O, pAMP18, pAMP18N, pAMPI, pAMPIC, pAMP 18NM and pAMP18B.

[0038]FIG. 2: M13, M13RT, pAMP18BH— and M13RTH−.

[0039]FIG. 3: pAMP1A.

[0040]FIG. 4: pDBH−Kpn, pDABH−, pDBH−KpnHis.

[0041]FIG. 5: pFastBac DUAL, pFastBac DUAL Nde, pDBH− and pDA.

[0042]FIG. 6: pDABH−His.

[0043] FIGS. 7-21 are more detailed maps of the plasmids described inFIGS. 1-6:

[0044]FIG. 7: pJD100.

[0045]FIG. 8: pAMP18N.

[0046]FIG. 9: pAMP1C.

[0047]FIG. 10: pAMP18NM.

[0048]FIG. 11: pAMP18B.

[0049]FIG. 12: M13RT.

[0050]FIG. 13: M13RTH−.

[0051]FIG. 14: pAMP18BH−.

[0052]FIG. 15: pDBH−.

[0053]FIG. 16: pAMP1A.

[0054]FIG. 17: pDBH−Kpn.

[0055]FIG. 18: pDBH−KpnHis.

[0056]FIG. 19: pDA.

[0057]FIG. 20: pDABH−.

[0058]FIG. 21: pDABH−His.

[0059] FIGS. 22-25 describe, in schematic form (with details omitted forclarity), the construction of the expression vector (pDAMVAH−BH—)whichplaces the AMV RT α and β genes under control of insect viral promoters:

[0060]FIG. 22: Cloning of AMV RT gene from RNA; pSPORT8.

[0061]FIG. 23: Construction of a His-tagged AMV RT β gene; pAMVN,pAMVNM, pAMVNMH−, pAMVC and pAMVBH−.

[0062]FIG. 24: Construction of clones for the AMV RT α subunit by PCR;pAMVA and pAMVAH−.

[0063]FIG. 25: Construction of vectors comprising the AMV RT α and βgenes; pD, pDAMVAH−, pDAMVA, pAMVH−BH−, pDAMVABH— and pJAMVBH−.

[0064] FIGS. 26-38 are more detailed maps of the plasmids described inFIGS. 22-25:

[0065]FIG. 26: pAMVN.

[0066]FIG. 27: pAMVC.

[0067]FIG. 28: pAMVNM.

[0068]FIG. 29: pAMVNMH−.

[0069]FIG. 30: pAMVBH−.

[0070]FIG. 31: pAMVA.

[0071]FIG. 32: pAMVAH−.

[0072]FIG. 33: pFastBacDual (pD).

[0073]FIG. 34: pDAMVA.

[0074]FIG. 35: pDAMVAH−.

[0075]FIG. 36: pDAMVABH−.

[0076]FIG. 37: pJAMVBH−.

[0077]FIG. 38: pDAMVAH−BH−.

[0078]FIG. 39 is a semi-logarithmic graph demonstrating RT activities ofvarious RTs incubated for the times and at the temperatures indicated.

[0079]FIG. 40 is an autoradiograph of cDNA products synthesized byvarious RTs at the temperatures (° C.) indicated from 1.4-, 2.4-, 4.4-and 7.5-Kb mRNAs over 50-minute reactions. M: ³²P-labeled lKb DNAladder.

[0080]FIG. 41 is an autoradiograph of cDNA products synthesized by RSVH⁻RT and SS II RT at the temperatures (° C.) indicated from 1.4-, 2.4-,4.4-, and 7.5-Kb mRNAs over 30-minute reactions. M: ³²P-labeled 1 Kb DNAladder.

[0081]FIG. 42 is a graph of the amounts of full length cDNA synthesizedby SSII and RSV H⁻ RT in FIG. 41 as a function of incubationtemperature.

[0082]FIG. 43 is a restriction map of plasmid pBP-RT (PCR).

[0083]FIG. 44 is a restriction map of plasmid pBP-RT (ATG).

[0084]FIG. 45 is a restriction map of plasmid pBK-RT15(ATG).

[0085]FIG. 46 is a restriction map of plasmid pFBBH−His.

[0086]FIG. 47 is a restriction map of plasmid pJB-His.

[0087]FIG. 48 is a restriction map of plasmid pJBD110E-His.

[0088]FIG. 49 is a restriction map of plasmid pDAD110E.

[0089]FIG. 50 is a restriction map of plasmid pFBBD110E-His.

[0090]FIG. 51 is a restriction map of plasmid pDABHis.

[0091]FIG. 52 is a restriction map of plasmid pDABD110EHis.

[0092]FIG. 53 is a restriction map of plasmid PDAD110EBHis.

[0093]FIG. 54 is a restriction map of plasmid pDAD110EBH110E.

DETAILED DESCRIPTION OF THE INVENTION

[0094] Overview

[0095] The present invention provides compositions and methods useful inovercoming the length limitations often observed during reversetranscription of nucleic acid molecules. Thus, the invention facilitatesthe production of full-length cDNA molecules not heretofore possible.

[0096] In general, the invention provides compositions for use inreverse transcription of a nucleic acid molecule comprising two or more,three or more, four or more, five or more, six or more, and the like,different polypeptides having reverse transcriptase activity. Thecompositions of the invention preferably comprise two to six, two tofive, two to four, two to three, and more preferably comprise two,polypeptides having reverse transcriptase activity. The enzymes in thesecompositions are preferably present in working concentrations and arereduced or substantially reduced in RNase H activity, although mixturesof enzymes, some having RNase H activity and some reduced orsubstantially reduced in RNase H activity, may be used in thecompositions of the invention. Alternatively, the reverse transcriptasesused in the compositions of the invention may have RNase H activity.Preferred reverse transcriptases include M-MLV H⁻ reverse transcriptase,RSV H⁻ reverse transcriptase, AMV H⁻ reverse transcriptase, RAV H⁻reverse transcriptase, MAV H⁻ reverse transcriptase and HIV H— reversetranscriptase or other ASLV H⁻ reverse transcriptases.

[0097] The invention is also directed to methods for reversetranscription of one or more nucleic acid molecules comprising mixingone or more nucleic acid templates, which is preferably RNA or messengerRNA (mRNA) and more preferably a population of mRNA molecules, with twoor more polypeptides having reverse transcriptase activity (or with thecompositions of the invention) and incubating the mixture underconditions sufficient to make a nucleic acid molecule or moleculescomplementary to all or a portion of the one or more templates. Suchnucleic acid synthesis may be accomplished by sequential or simultaneousor separate addition of multiple reverse transcriptases. Preferably, twoor more, three or more, four or more, five or more, six or more, and thelike, reverse transcriptases are used, or a range of two to six, two tofive, two to four, two to three and more preferably two, reversetranscriptases are used. To make the nucleic acid molecule or moleculescomplementary to the one or more templates, a primer (e.g., an oligo(dT) primer) and one or more nucleotides are used for nucleic acidsynthesis in the 3′ to 5′ direction. Nucleic acid molecules suitable forreverse transcription according to this aspect of the invention includeany nucleic acid molecule, particularly those derived from a prokaryoticor eukaryotic cell. Such cells may include normal cells, diseased cells,transformed cells, established cells, progenitor cells, precursor cells,fetal cells, embryonic cells, bacterial cells, yeast cells, animal cells(including human cells), avian cells, plant cells and the like, ortissue isolated from a plant or an animal (e.g., human, cow, pig, mouse,sheep, horse, monkey, canine, feline, rat, rabbit, bird, fish, insect,etc.). Such nucleic acid molecules may also be isolated from viruses.

[0098] The invention further provides methods for amplifying orsequencing a nucleic acid molecule comprising contacting the nucleicacid molecule with two or more polypeptides having reverse transcriptaseactivity (or with the compositions of the invention). Such reactions maybe accomplished by sequential or simultaneous or separate addition ofthe two or more polypeptides having reverse transcriptase activity tothe reaction mixtures. Preferred such methods comprise one or morepolymerase chain reactions (PCRs).

[0099] The invention also provides cDNA molecules or amplified nucleicacid molecules produced according to the above-described methods,vectors (particularly expression vectors) comprising these cDNAmolecules or amplified nucleic acid molecules, and recombinant hostcells comprising such cDNA molecules, amplified nucleic acid moleculesor vectors. The invention also provides methods of producing apolypeptide comprising culturing these recombinant host cells andisolating the polypeptide, and provides a polypeptide produced by suchmethods.

[0100] The invention also provides kits for use in accordance with theinvention. Such kits comprise a carrier means, such as a box or carton,having in close confinement therein one or more container means, such asvials, tubes, bottles and the like, wherein the kit comprises, in thesame or different containers, two or more polypeptides having reversetranscriptase activity. The kits of the invention may also comprise, inthe same or different containers, one or more DNA polymerases, asuitable buffer and/or one or more nucleotides (such as deoxynucleosidetriphosphates (dNTPs)).

[0101] The invention also concerns a substantially pure RSV reversetranscriptase (RSV RT), which may or may not be reduced or substantiallyreduced in RNase H activity. Such RSV RTs may comprise one or moresubunits (or derivatives, variants, fragments or mutants thereof)selected from one or more a subunits, one or more β subunits, and one ormore βp4 subunits, any or all of which may or may not be reduced orsubstantially reduced in RNase H activity. In one preferred aspect ofthe invention, the RSV RT may comprise an α subunit reduced orsubstantially reduced in RNase H activity and a β subunit having RNase Hactivity (i.e., RSV αH⁻βH⁺ RT). In a preferred aspect of thisembodiment, the gene encoding the a subunit has been modified or mutatedto reduce RNase H activity while the gene encoding the β subunit has notbeen mutated or modified in this manner. Such mutations or modificationsare preferably made within the RNase H domain of the a subunit.Unexpectedly, the phenotype of this construct showed substantiallyreduced (i.e., approximately 5% of wildtype) RNase H activity. Inanother preferred aspect, the RSV RT may comprise an a subunit reducedor substantially reduced in RNase H activity and a β subunit alsoreduced or substantially reduced in RNase H activity (i.e., RSV αH⁻/βH⁻RT). In another preferred aspect, the RSV RT may comprise two βsubunits, either or both of which may or may not be reduced orsubstantially reduced in RNAse H activity (i.e., RSV βH⁻/H⁻RT; RSVβH⁻/βH⁺ RT; or RSV βH⁺/βH⁺ RT). In another preferred aspect, the RSV RTmay comprise a single a subunit, which may or may not be reduced orsubstantially reduced in RNAse H activity (i.e., RSV αH⁻ RT or RSV αH⁺RT). In another preferred aspect, the RSV RT may comprise one or moreβp4 subunits, any or all of which may or may not be reduced orsubstantially reduced in RNAse H activity (e.g., RSV βp4H⁻/βp4H⁻ RT; RSVβp4H⁻/βp4H⁺ RT; RSV βp4H⁺/β4H⁺ RT; RSV αH⁻/βp4H⁺ RT; RSV αH⁺/βp4H⁺ RT;RSV αH⁻/βp4H⁻ RT; RSV αH⁺/βp4H⁻ RT; RSV βH⁻/βp4H⁺ RT; RSV βH⁻/βp4H⁻ RT;RSV βH⁺/βp4H⁻ RT; RSV βH ⁺/βp4H⁺ RT; etc.). As will be recognized,derivatives, variants, fragments or mutants of any or all of the abovesubunits may also be used in accordance with the invention.

[0102] In a related aspect of the invention, where the RSV RTs comprisetwo or more subunits (or derivatives, variants, fragments or mutantsthereof) and preferably comprise two subunits (e.g., a dimer), at leastone but preferably not all of these subunits may be modified or mutatedto reduce, substantially reduce or eliminate the polymerase activity ofat least one subunit (e.g., pol-). In a preferred aspect, for an RSV RTwhich comprises an α and β subunit, the β subunit has been modified ormutated (preferably by recombinant techniques) to reduce, substantiallyreduce or eliminate the polymerase activity while the polymeraseactivity of the a subunit has not been mutated or modified in thismanner. Preferably, the a subunit of such RSV RT has also been modifiedor mutated to reduce or substantially reduce RNase H activity while theβ subunit has not been mutated or modified in this manner. Such aconstruct may be designated αH−/βH+pol-. Any number of combinations ofsubunits can be prepared in which mutations or modifications are made inone subunit of a two subunit enzyme, and these constructs may becombined with other modifications or mutations, such as those whichreduce or substantially reduce RNase H activity. Illustrated examplesinclude but are not limited to RSV αH−/pH−pol-; RSV αH+/βH+ pol-; RSVαH− pol-/βH+; RSV αH− pol-/βH−; RSV αH⁺ pol-/βH⁺; RSV αH−/βH+ pol-; RSVβH−/βH+ pol-; RSV βH+/βH+ pol-; RSV βp4H−/βp4H− pol-; RSV βp4H−/βp4H+pol-; RSV βp4H+/βp4H+ pol-; RSV αH−/βp4H− pol-; RSV αH+/βp4H+pol-; RSVαH+/βp4H− pol-; RSV βH−/βp4H+ pol-; RSV βH−/βp4H+pol-; RSV βH+/βp4H−pol-; etc. In a preferred aspect, the polymerase domain of the subunitis modified or mutated (one or more point mutations, deletion mutationsand/or insertion mutations) by recombinant techniques. In anotheraspect, the nucleotide binding site of the polymerase domain is modifiedor mutated. In a preferred aspect, one or more acidic amino acids withinthe nucleotide binding site are substituted with different amino acids.Particularly preferred amino acids within the nucleotide binding sitefor mutation or modification include, but are not limited to, Asp¹⁰⁷,Leu¹⁰⁸, Lys¹⁰⁹, and Asp¹¹⁰, or the corresponding amino acid sequence.

[0103] The invention also relates to methods of producing the RSV RTs ofthe invention, which methods comprise obtaining a host cell comprising anucleic acid sequence encoding one or more a subunits (or derivatives,variants, fragments or mutants thereof) and/or a nucleic acid sequenceencoding one or more β subunits (or derivatives, variants, fragments ormutants thereof) and/or a nucleic acid sequence encoding one or more βp4subunits (or derivatives, variants, fragments or mutants thereof), andculturing the host cell under conditions sufficient to produce the RSVRTs of the invention. The nucleic acid sequences encoding such αsubunit(s) and/or such β subunit(s) and/or such βp4 subunit(s) may becontained in the same vector or in different vectors. In accordance withthe invention, such α subunit(s) and/or β subunit(s) and/or βp4subunit(s) maybe produced separately and mixed before or after isolationof each subunit to form the RSV RTs of the invention. Alternatively,such a and/or β subunits and/or βp4 subunits may be expressedsimultaneously (i.e., co-expressed) in the same host cell, therebyproducing an RSV RT comprising an α and a β subunit, an a subunit alone,a β subunit alone, a βp4 subunit alone, a β subunit and a βp4 subunit,two β subunits, or two βp4 subunits, or derivatives, variants, fragmentsor mutants thereof In a preferred aspect, the α subunit (or derivatives,variants, fragments or mutants thereof) is simultaneously expressed in ahost cell with the β subunit (or derivatives, variants, fragments ormutants thereof). In a related aspect of the invention, the β or βp4subunits (or derivatives, fragments or mutants thereof) may be expressedin a host or host cell which accomplishes in vivo processing of some orall of such β or βp4 subunits to form the corresponding a subunit. Thepresence of both the β and the α subunits allows in vivo formation of anRSV RT which comprises an α and a β subunit. Such in vivo processing ispreferably accomplished by expressing the β and/or βp4 subunits (orderivatives, variants, fragments or mutants thereof) in a host cell,which may be prokaryotic or eukaryotic, having appropriate processingenzymes or proteins which cleave such β or βp4 subunits to form acorresponding a subunit. Such processing enzymes or proteins may beintroduced and expressed in the host system by recombinant means or mayexist naturally in the host system. Preferred hosts for in vivoprocessing include eukaryotic cells or organelles such as yeast, fungi,plants, animals, insects, fish, and the like. Recombinant systems(vectors, expression vectors, promoters, etc.) which allow cloning ofthe β or βp4 subunits (or derivatives, fragments or mutants thereof) forin vivo processing are well known to one of ordinary skill in the art.As noted above, any or all of the α and/or β and/or βp4 subunits of theRSV RT (or derivatives, variants, fragments or mutants thereof) producedby these recombinant techniques may be reduced or substantially reducedin RNase H activity.

[0104] These RSV RTs or subunits thereof may then be isolated from thehost cell, and may be substantially purified by any method of proteinpurification that will be familiar to those of ordinary skill in the art(e.g., chromatography, electrophoresis, dialysis, high-saltprecipitation, or combinations thereof). The invention also relates tokits comprising one or more of the RSV RTs of the invention.

[0105] The invention also concerns a substantially pure AvianMyeloblastosis Virus reverse transcriptase (AMV RT), which may or maynot be reduced or substantially reduced in RNase H activity. Such AMVRTs may comprise one or more subunits selected from one or more asubunits, one or more β subunits, and one or more βp4 subunits (orderivatives, variants, fragments or mutants thereof), any or all ofwhich may or may not be reduced or substantially reduced in RNase Hactivity. In one preferred aspect of the invention, the AMV RT maycomprise an a subunit reduced or substantially reduced in RNase Hactivity and a β subunit having RNase H activity (i.e., AMV αH⁻ βH⁺ RT).In a particularly preferred aspect of this embodiment, the gene encodingthe α subunit has been modified or mutated to reduce RNase activity(preferably within the RNase H domain) while the gene encoding the βsubunit has not been modified or mutated to affect RNase H activity.Unexpectedly, this construct demonstrates a phenotype in which the RNaseH activity of the AMV RT comprising the a subunit and the β subunit issubstantially reduced in RNase H activity (i.e., approximately 5% ofwildtype). In another preferred aspect, the AMV RT may comprise an asubunit reduced or substantially reduced in RNase H activity and a βsubunit also reduced or substantially reduced in RNase H activity (i.e.,AMV αH⁻/βH⁻ RT). In another preferred aspect, the AMV RT may comprisetwo β subunits, either or both of which may or may not be reduced orsubstantially reduced in RNAse H activity (i.e., AMV βH⁻/βH⁻ RT; AMVβH⁻/βH⁺ RT; or AMV βH⁺/βH⁺ RT). In another preferred aspect, the AMV RTmay comprise a single α subunit, which may or may not be reduced orsubstantially reduced in RNAse H activity (i.e., AMV αH⁻ RT or AMV αH⁺RT). In another preferred aspect, the AMV RT may comprise one or moreβp4 subunits, any or all of which may or may not be reduced orsubstantially reduced in RNAse H activity (e.g., AMV βp4H⁻/βp4H⁻ RT; AMVβp4H⁻/βp4H⁺ RT; AMV βp4H⁺/βp4H⁺ RT; AMV αH⁻/βp4H⁺ RT; AMV αH⁺/βp4H⁺ RT;AMV αH⁻/βp4H⁻ RT; AMV αH⁺/βp4H⁻ RT; AMV βH⁻/βp4H⁻ RT; AMV βH⁺/βp4H⁻ RT;etc.).

[0106] In a related aspect of the invention, where the AMV RTs comprisetwo or more subunits (or derivatives, variants, fragments or mutantsthereof) and preferably comprise two subunits (e.g., a dimer), at leastone but preferably not all of these subunits may be modified or mutatedto reduce, substantially reduce or eliminate the polymerase activity ofat least one subunit (e.g., pol-). In a preferred aspect, for an AMV RTwhich comprises an α and β subunit, the β subunit has been modified ormutated (preferably by recombinant techniques) to reduce, substantiallyreduce or eliminate the polymerase activity while the polymeraseactivity of the α subunit has not been mutated or modified in thismanner. Preferably, the α subunit of such AMV RT has also been modifiedor mutated to reduce or substantially reduce RNase H activity while theβ subunit has not been mutated or modified in this manner. Such aconstruct may be designated αH−/H+pol-. Any number of combinations ofsubunits can be prepared in which mutations or modifications are made inone subunit of a two subunit enzyme, and these constructs may becombined with other modifications or mutations, such as those whichreduce or substantially reduce RNase H activity. Illustrated examplesinclude but are not limited to AMV αH−/βH− pol-; AMV αH+/βH+ pol-; AMVαH− pol-/βH+; AMV αH− pol-/βH−; AMV αH+ pol-/PH+; AMV βH−/βH− pol-; AMVβH−/βH+ pol-; AMV βH+/βH+ pol-; AMV βp4H−/βp4H− pol-; AMV βp4H−/βp4H+pol-; AMV βp4H+/βp4H+ pol-; AMV αH−/βp4H− pol-; AMV αH+/βp4H+ pol-; AMVαH+/βp4H− pol-; AMV βH−/βp4H+ pol-; AMV βH−/βp4H+ pol-; AMV βH+/βp4H−pol-; etc. In a preferred aspect, the polymerase domain of the subunitis modified or mutated (one or more point mutations, deletion mutationsand/or insertion mutations) by recombinant techniques. In anotheraspect, the nucleotide binding site of the polymerase domain is modifiedor mutated. In a preferred aspect, one or more acidic amino acids withinthe nucleotide binding site are substituted with different amino acids.Particularly preferred amino acids within the nucleotide binding sitefor mutation or modification include, but are not limited to, Asp¹⁰⁷,Leu¹⁰⁸, Lys¹⁰⁹, and Asp¹¹⁰, or the corresponding amino acid sequence.

[0107] The invention also relates to methods of producing the AMV RTs ofthe invention, which methods comprise obtaining a host cell comprising anucleic acid sequence encoding one or more a subunits (or derivatives,variants, fragments or mutants thereof) and/or a nucleic acid sequenceencoding one or more β subunits (or derivatives, variants, fragments ormutants thereof) and/or a nucleic acid sequence encoding one or more βp4subunits (or derivatives, variants, fragments or mutants thereof), andculturing the host cell under conditions sufficient to produce the AMVRTs. The nucleic acid sequences encoding such a subunit(s) and/or such βsubunit(s) and/or such βp4 subunit(s) may be contained in the samevector or in different vectors. In accordance with the invention, such asubunit(s) and/or β subunit(s) and/or βp4 subunit(s) may be producedseparately and mixed before or after isolation of each subunit to formthe AMV RTs of the invention. Alternatively, such α and/or β and/or βp4subunits may be expressed simultaneously (i.e., co-expressed) in thesame host cell, thereby producing an AMV RT comprising an α and a βsubunit, an α subunit alone, a β subunit alone, a βp4 subunit alone, a βsubunit and a βp4 subunit, two β subunits, or two βp4 subunits, orderivatives, variants, fragments or mutants thereof In a preferredaspect, the a subunit is simultaneously expressed in a host cell withthe β subunit. In a related aspect of the invention, the β or βp4subunits (or derivatives, variants, fragments or mutants thereof) may beexpressed in a host or host cell which accomplishes in vivo processingas described above for production of RSV RTs. Thus, by expressing a βsubunit and/or a βp4 subunit (or derivatives, fragments or mutantsthereof) in a host system which has appropriate processing enzymes orproteins, the corresponding α subunit may be produced allowing formationof an AMV RT which comprises an α and a β subunit, an α and a βp4subunit, etc. As noted above, any or all of the α and/or β and/or βp4subunits (or derivatives, variants, fragments or mutants thereof) of theAMV RT may be reduced or substantially reduced in RNase H activity.These AMV RTs or subunits thereof may then be isolated from the hostcell and may be substantially purified by those methods described abovefor purification of RSV RTs. The invention also relates to kitscomprising one or more of the AMV RTs of the invention.

[0108] The invention also relates to other substantially pure ASLVreverse transcriptases, which may or may not be reduced or substantiallyreduced in RNase H activity. Such ASLV RTs may comprise one or moresubunits (or derivatives, variants, fragments or mutants thereof)selected from one or more α subunits, one or more β subunits, and one ormore βp4 subunits, as described for RSV RT and AMV RT above. Theinvention also relates to methods of producing such ASLV RTs of theinvention as described above for RSV RT and AMV RT.

[0109] The invention also concerns the RSV reverse transcriptases andAMV reverse transcriptases or other ASLV reverse transcriptases andsubunits thereof (and derivatives, variants, fragments and mutantsthereof) of the invention which have functional activity, as measured bythe ability of the proteins to produce first strand cDNA from a mRNAtemplate. Such functional activity may be measured in accordance withthe invention based on the total full-length reverse transcribed productmade during the synthesis reaction. The amount of product is preferablymeasured based on the mass (e.g, nanograms) of products produced,although other means of measuring the amount of product will berecognized by one of ordinary skill in the art. Additionally, functionalactivity may be measured in terms of the percentage of full-lengthproducts produced during a cDNA synthesis reaction. For example, thepercent full-length functional activity may be determined by dividingthe amount of full-length product by the amount of total productproduced during a cDNA synthesis reaction and multiplying the result by100 to obtain the percentage. The RSV and AMV reverse transcriptases andtheir subunits (and derivatives, variants, fragments and mutantsthereof) of the invention produce greater than about 4%, preferablygreaterthan about 5%, more preferably greater than about 7.5%, stillmore preferably greater than about 10%, still more preferably greaterthan about 20%, and most preferably greater than about 25%, full-lengthcDNA in a nucleic acid synthesis reaction. Preferred ranges of suchpercentages include about 5% to about 100%, about 7.5% to about 75%,about 7.5% to about 50%, about 10% to about 50%, about 15% to about 40%,about 20% to about 40%, and about 20% to about 50%. Functional activitymay also be measured in accordance with the invention by determining thepercentage of total cDNA product compared to the amount of input mRNA inthe synthesis reaction. Thus, the total amount of cDNA product isdivided by the amount of input mRNA, the result of which is multipliedby 100 to determine the percentage functional activity associated withamount of product produced compared to amount of the template used.Preferably, the reverse transcriptases of the invention produce greaterthan about 15%, more preferably greater than about 20%, still morepreferably greater than about 25%, still more preferably greater thanabout 30%, and most preferably greater than about 40%, of cDNA comparedto input mRNA in the cDNA synthesis reaction. Preferred ranges of suchpercentages include about 5% to about 100%, about 10% to about 80%,about 15% to about 80%, about 15% to about 75%, about 20% to about 75%,about 20% to about 70%, about 25% to about 75%, about 25% to about 70%,about 25% to about 60%, and about 25% to about 50%. The AMV and RSVreverse transcriptases and their subunits (and derivatives, variants,fragments and mutants thereof) of the invention preferably have specificactivities greater than about 5 units/mg, more preferably greater thanabout 50 units/mg, still more preferably greater than about 100units/mg, 250 units/mg, 500 units/mg, 1000 units/mg, 5000 units/mg or10,000 units/mg, and most preferably greater than about 15,000 units/mg,greater than about 16,000 units/mg, greater than about 17,000 units/mg,greater than about 18,000 units/mg, greater than about 19,000 units/mgand greater than about 20,000 units/mg. Preferred ranges of specificactivities for the AMV and RSV RTs and their subunits (or derivatives,variants, fragments or mutants thereof) of the invention include aspecific activity from about 5 units/mg to about 140,000 units/mg, aspecific activity from about 5 units/mg to about 125,000 units/mg, aspecific activity of from about 50 units/mg to about 100,000 units/mg, aspecific activity from about 100 units/mg to about 100,000 units/mg, aspecific activity from about 250 units/mg to about 100,000 units/mg, aspecific activity from about 500 units/mg to about 100,000 units/mg, aspecific activity from about 1000 units/mg to about 100,000 units/mg, aspecific activity from about 5000 units/mg to about 100,000 units/mg, aspecific activity from about 10,000 units/mg to about 100,000 units/mg,a specific activity from about 25,000 units/mg to about 75,000 units/mg.Other preferred ranges of specific activities include a specificactivity of from about 20,000 units/mg to about 140,000 units/mg, aspecific activity from about 20,000 units/mg to about 130,000 units/mg,a specific activity from about 20,000 units/mg to about 120,000units/mg, a specific activity from about 20,000 units/mg to about110,000 units/mg, a specific activity from about 20,000 units/mg toabout 100,000 units/mg, a specific activity from about 20,000 units/mgto about 90,000 units/mg, a specific activity from about 25,000 units/mgto about 140,000 units/mg, a specific activity from about 25,000units/mg to about 130,000 units/mg, a specific activity from about25,000 units/mg to about 120,000 units/mg, a specific activity fromabout 25,000 units/mg to about 110,000 units/mg, a specific activityfrom about 25,000 units/mg to about 100,000 units/mg, and a specificactivity from about 25,000 units/mg to about 90,000 units/mg.Preferably, the lower end of the specific activity range may vary from30,000, 35,000, 40,000, 45,000, 50,000, 5,000, 60,000, 65,000, 70,000,75,000, and 80,000 units/mg, while the upper end of the range may varyfrom 150,000, 140,000, 130,000, 120,000, 110,000, 100,000, and 90,000units/mg. In accordance with the invention, specific activity is ameasurement of the enzymatic activity (in units) of the protein orenzyme relative to the total amount of protein or enzyme used in areaction. The measurement of a specific activity may be determined bystandard techniques well-known to one of ordinary skill in the art.Preferred assays for determining the specific activity of an enzyme orprotein are described in detail in the Examples below.

[0110] The RSV RTs and AMV RTs or other ASLV RTs and their subunits (orderivatives, variants, fragments or mutants thereof) of the inventionmay be used to make nucleic acid molecules from one or more templates.Such methods comprise mixing one or more nucleic acid templates (e.g.,mRNA, and more preferably a population of mRNA molecules) with one ormore of the RSV RTs and/or one or more AMV RTs and/or other ASLV RTs ofthe invention and incubating the mixture under conditions sufficient tomake one or more nucleic acid molecules complementary to all or aportion of the one or more nucleic acid templates.

[0111] The invention also relates to methods for the amplification ofone or more nucleic acid molecules comprising mixing one or more nucleicacid templates with one or more of the RSV RTs and/or one or more of theAMV RTs and/or other ASLV RTs of the invention and optionally with oneor more DNA polymerases, and incubating the mixture under conditionssufficient to amplify one or more nucleic acid molecules complementaryto all or a portion of the one or more nucleic acid templates.

[0112] The invention also concerns methods for the sequencing of one ormore nucleic acid molecules comprising (a) mixing one or more nucleicacid molecules to be sequenced with one or more primer nucleic acidmolecules, one or more RSV RTs and/or one or more AMV RTs and/or otherASLV RTs of the invention, one or more nucleotides and one or moreterminating agents; (b) incubating the mixture under conditionssufficient to synthesize a population of nucleic acid moleculescomplementary to all or a portion of the one or more nucleic acidmolecules to be sequenced; and (c) separating the population of nucleicacid molecules to determine the nucleotide sequence of all or a portionof the one or more nucleic acid molecules to be sequenced.

[0113] The invention also concerns methods for elevated- orhigh-temperature reverse transcription of a nucleic acid moleculecomprising (a) mixing a nucleic acid template (preferably an RNA (e.g.,a mRNA molecule or a polyA+ RNA molecule) or a DNA molecule) with one ormore polypeptides having reverse transcriptase activity; and (b)incubating the mixture at a temperature of 50° C. or greater and underconditions sufficient to make a first nucleic acid molecule (such as afull length cDNA molecule) complementary to all or a portion of thenucleic acid template. In preferred such methods, the mixture isincubated at a temperature of about 51° C. or greater, about 52° C. orgreater, about 53° C. or greater, about 54° C. or greater, about 55° C.or greater, about 56° C. or greater, about 57° C. or greater, about 58°C. orgreater, about 59° C. orgreater, about 60° C. or greater, about 61°C. or greater, about 62° C. or greater; or at a temperature range offrom about 50° C. to about 70° C., about 51° C. to about 70° C., about52° C. to about 70° C., about 53° C. to about 70° C., about 54° C. toabout 70° C., about 55° C. to about 70° C., about 55° C. to about 65°C., about 56° C. to about 65° C., about 56° C. to about 64° C. or about56° C. to about 62° C. The invention is also directed to such methodswhich further comprise incubating the first nucleic acid molecule underconditions sufficient to make a second nucleic acid moleculecomplementary to all or portion of the first nucleic acid molecule.According to the invention, the first and second nucleic acid moleculesproduced by these methods may be DNA molecules, and may form a doublestranded DNA molecule which may be a full length cDNA molecule. The oneor more polypeptides having reverse transcriptase activity that are usedin these methods preferably are reduced or substantially reduced inRNase H activity, and may be selected from the group consisting of oneor more subunits of AMV reverse transcriptase and one or more subunitsof RSV reverse transcriptase and one or more subunits of other ASLVreverse transcriptases (or derivatives, variants, fragments or mutantsthereof). As noted above, such AMV RTs or RSV RTs or other ASLV RTs maycomprise one or more α subunits, one or more β subunits, and/or one ormore βp4 subunits, any or all of which subunits may be reduced orsubstantially reduced in RNase H activity. Particularly preferredpolymerases having RT activity for use in these methods are those RSVRTs and AMV RTs or other ASLV RTs and their subunits (or derivatives,variants, fragments or mutants thereof) described above.

[0114] The invention also concerns nucleic acid molecules produced bysuch methods (which may be full-length cDNA molecules), vectors(particularly expression vectors) comprising these nucleic acidmolecules and host cells comprising these vectors and nucleic acidmolecules.

[0115] Sources of Enzymes

[0116] Enzymes for use in the compositions, methods and kits of theinvention include any enzyme having reverse transcriptase activity. Suchenzymes include, but are not limited to, retroviral reversetranscriptase, retrotransposon reverse transcriptase, hepatitis Breverse transcriptase, cauliflower mosaic virus reverse transcriptase,bacterial reverse transcriptase, Tth DNA polymerase, Taq DNA polymerase(Saiki, R. K., et al., Science 239:487-491 (1988); U.S. Pat. Nos.4,889,818 and 4,965,188), The DNA polymerase (WO 96/10640), Tma DNApolymerase (U.S. Pat. No. 5,374,553) and mutants, fragments, variants orderivatives thereof (see, e.g., commonly owned, co-pending U.S. patentapplication Ser. Nos. 08/706,702 and 08/706,706, both filed Sep. 9,1996, which are incorporated by reference herein in their entireties).As will be understood by one of ordinary skill in the art, modifiedreverse transcriptases may be obtained by recombinant or geneticengineering techniques that are routine and well-known in the art.Mutant reverse transcriptases can, for example, be obtained by mutatingthe gene or genes encoding the reverse transcriptase of interest bysite-directed or random mutagenesis. Such mutations may include pointmutations, deletion mutations and insertional mutations. Preferably, oneor more point mutations (e.g., substitution of one or more amino acidswith one or more different amino acids) are used to construct mutantreverse transcriptases of the invention. Fragments of reversetranscriptases may be obtained by deletion mutation by recombinanttechniques that are routine and well-known in the art, or by enzymaticdigestion of the reverse transcriptase(s) of interest using any of anumber of well-known proteolytic enzymes.

[0117] Preferred enzymes for use in the invention include those that arereduced or substantially reduced in RNase H activity. Such enzymes thatare reduced or substantially reduced in RNase H activity may be obtainedby mutating the RNase H domain within the reverse transcriptase ofinterest, preferably by one or more point mutations, one or moredeletion mutations, and/or one or more insertion mutations as describedabove. By an enzyme “substantially reduced in RNase H activity” is meantthat the enzyme has less than about 30%, less than about 25%, 20%, morepreferably less than about 15%, less than about 10%, less than about7.5%, or less than about 5%, and most preferably less than about 5% orless than about 2%, of the RNase H activity of the correspondingwildtype or RNase H+enzyme such as wildtype Moloney Murine LeukemiaVirus (M-MLV), Avian Myeloblastosis Virus (AMV) or Rous Sarcoma Virus(RSV) reverse transcriptases. The RNase H activity of any enzyme may bedetermined by a variety of assays, such as those described, for example,in U.S. Pat. No. 5,244,797, in Kotewicz, M. L., et al., Nucl. Acids Res.16:265 (1988), in Gerard, G. F., et al., FOCUS 14(5):91 (1992), and inU.S. Pat. No. 5,668,005, the disclosures of all of which are fullyincorporated herein by reference.

[0118] Particularly preferred enzymes for use in the invention include,but are not limited to, M-MLV H⁻ reverse transcriptase, RSV H⁻ reversetranscriptase, AMV H⁻ reverse transcriptase, RAV H⁻ reversetranscriptase, MAV H⁻ reverse transcriptase and HIV H⁻ reversetranscriptase. It will be understood by one of ordinary skill, however,that any enzyme capable of producing a DNA molecule from a ribonucleicacid molecule (i.e., having reverse transcriptase activity) that issubstantially reduced in RNase H activity may be equivalently used inthe compositions, methods and kits of the invention.

[0119] Enzymes used in the invention may have distinct reversetranscription pause sites with respect to the template nucleic acid.Whether or not two enzymes have distinct reverse transcription pausesites may be determined by a variety of assays, including, for example,electrophoretic analysis of the chain lengths of DNA molecules producedby the two enzymes (Weaver, D. T., and DePamphilis, M. L., J. Biol.Chem. 257(4):2075-2086 (1982); Abbots, J., et al., J. Biol. Chem.268(14): 10312-10323 (1993)), or by other assays that will be familiarto one of ordinary skill in the art. As described above, these distincttranscription pause sites may represent secondary structural andsequence barriers in the nucleic acid template which occur frequently athomopolymer stretches. Thus, for example, the second enzyme may reversetranscribe to a point (e.g., a hairpin) on the template nucleic acidthat is proximal or distal (i.e., 3′ or 5′) to the point to which thefirst enzyme reverse transcribes the template nucleic acid. Thiscombination of two or more enzymes having distinct reverse transcriptionpause sites facilitates production of full-length cDNA molecules sincethe secondary structural and sequence barriers may be overcome.Moreover, the elevated- or high-temperature reverse transcription of theinvention may also assist in overcoming secondary structural andsequence barriers during nucleic acid synthesis. Thus, the elevated- orhigh-temperature synthesis may be used in combination with the two ormore reverse transcriptases (preferably using an AMV RT, an RSV RT orother ASLV RT) to facilitate full-length cDNA synthesis.

[0120] A variety of DNA polymerases are useful in accordance with thepresent invention. Such polymerases include, but are not limited to,Thermus thermophilus (Tth) DNA polymerase, Thermus aquaticus (Taq) DNApolymerase, Thermotoga neapolitana (Tne) DNA polymerase, Thermotogamaritima (Tma) DNA polymerase, Thermococcus litoralis (Tli or VENT™) DNApolymerase, Pyrococcus furiosis (Pfu) DNA polymerase, DEEPVEN™ DNApolymerase, Pyrococcus woosii (Pwo) DNA polymerase, Bacillussterothermophilus (Bst) DNA polymerase, Bacillus caldophilus (Bca) DNApolymerase, Sulfolobus acidocaldarius (Sac) DNA polymerase, Thermoplasmaacidophilum (Tac) DNA polymerase, Thermus flavus (Tfl/Tub) DNApolymerase, Thermus ruber (Tru) DNA polymerase, Thermus brockianus(DYNAZYME™) DNA polymerase, Methanobacterium thermoautotrophicum (Mth)DNA polymerase, Mycobacterium spp. DNA polymerase (Mtb, Mlep), andmutants, variants and derivatives thereof.

[0121] DNA polymerases used in accordance with the invention may be anyenzyme that can synthesize a DNA molecule from a nucleic acid template,typically in the 5′ to 3′ direction. Such polymerases may be mesophilicor thermophilic, but are preferably thermophilic. Mesophilic polymerasesinclude T5 DNA polymerase, T7 DNA polymerase, Klenow fragment DNApolymerase, DNA polymerase III, and the like. Preferred DNA polymerasesare thermostable DNA polymerases such as Taq, Tne, Tma, Pfu, VENT™,DEEPVENT™, Tth and mutants, variants and derivatives thereof (U.S. Pat.No. 5,436,149; U.S. Pat. No. 5,512,462; WO 92/06188; WO 92/06200; WO96/10640; Barnes, W. M., Gene 112:29-35 (1992); Lawyer, F. C., et al.,PCR Meth. Appl. 2:275-287 (1993); Flaman, J.-M., et al., Nucl. AcidsRes. 22(15):3259-3260 (1994)). For amplification of long nucleic acidmolecules (e.g., nucleic acid molecules longer than about 3-5 Kb inlength), at least two DNA polymerases (one substantially lacking 3′exonuclease activity and the other having 3′ exonuclease activity) aretypically used. See U.S. Pat. No. 5,436,149; U.S. Pat. No. 5,512,462;Barnes, W. M., Gene 112:29-35 (1992); and commonly owned, co-pendingU.S. patent application Ser. No. 08/801,720, filed Feb. 14, 1997, thedisclosures of all of which are incorporated herein in their entireties.Examples of DNA polymerases substantially lacking in 3′ exonucleaseactivity include, but are not limited to, Taq, Tne(exo⁻), Tma,Pfu(exo⁻), Pwo and Tth DNA polymerases, and mutants, variants andderivatives thereof Nonlimiting examples of DNA polymerases having 3′exonuclease activity include Pfu/DEEPVENT™ and Tli/VENT™ and mutants,variants and derivatives thereof Polypeptides having reversetranscriptase activity for use in the invention may be obtainedcommercially, for example from Life Technologies, Inc. (Rockville, Md.),Pharmacia (Piscataway, N.J.), Sigma (Saint Louis, Mo.) or BoehringerMannheim Biochemicals (Indianapolis, Ind.). Alternatively, polypeptideshaving reverse transcriptase activity may be isolated from their naturalviral or bacterial sources according to standard procedures forisolating and purifying natural proteins that are well-known to one ofordinary skill in the art (see, e.g., Houts, G. E., et al., J. Virol.29:517 (1979)). In addition, the polypeptides having reversetranscriptase activity may be prepared by recombinant DNA techniquesthat are familiar to one of ordinary skill in the art (see, e.g.,Kotewicz, M. L., et al., Nucl. Acids Res. 16:265 (1988); Soltis, D. A.,and Skalka, A. M., Proc. Natl. Acad. Sci. USA 85:3372-3376 (1988)).

[0122] DNA polymerases for use in the invention may be obtainedcommercially, for example from Life Technologies, Inc. (Rockville, Md.),Perkin-Elmer (Branchburg, New Jersey), New England BioLabs (Beverly,Mass.) or Boehringer Mannheim Biochemicals (Indianapolis, Ind.).

[0123] Formulation of Enzyme Compositions

[0124] To form the compositions of the present invention, two or morereverse transcriptases are preferably admixed in a buffered saltsolution. One or more DNA polymerases and/or one or more nucleotides mayoptionally be added to make the compositions of the invention. Morepreferably, the enzymes are provided at working concentrations in stablebuffered salt solutions. The terms “stable” and “stability” as usedherein generally mean the retention by a composition, such as an enzymecomposition, of at least 70%, preferably at least 80%, and mostpreferably at least 90%, of the original enzymatic activity (in units)after the enzyme or composition containing the enzyme has been storedfor about one week at a temperature of about 4° C., about two to sixmonths at a temperature of about −20° C., and about six months or longerat a temperature of about −80° C. As used herein, the term “workingconcentration” means the concentration of an enzyme that is at or nearthe optimal concentration used in a solution to perform a particularfunction (such as reverse transcription of nucleic acids).

[0125] The water used in forming the compositions of the presentinvention is preferably distilled, deionized and sterile filtered(through a 0.1-0.2 micrometer filter), and is free of contamination byDNase and RNase enzymes. Such water is available commercially, forexample from Sigma Chemical Company (Saint Louis, Mo.), or may be madeas needed according to methods well known to those skilled in the art.

[0126] In addition to the enzyme components, the present compositionspreferably comprise one or more buffers and cofactors necessary forsynthesis of a nucleic acid molecule such as a cDNA molecule.Particularly preferred buffers for use in forming the presentcompositions are the acetate, sulfate, hydrochloride, phosphate or freeacid forms of Tris-(hydroxymethyl)aminomethane (TRIS®), althoughalternative buffers of the same approximate ionic strength and pKa asTRIS® may be used with equivalent results. In addition to the buffersalts, cofactor salts such as those of potassium (preferably potassiumchloride or potassium acetate) and magnesium (preferably magnesiumchloride or magnesium acetate) are included in the compositions.Addition of one or more carbohydrates and/or sugars to the compositionsand/or synthesis reaction mixtures may also be advantageous, to supportenhanced stability of the compositions and/or reaction mixtures uponstorage. Preferred such carbohydrates or sugars for inclusion in thecompositions and/or synthesis reaction mixtures of the inventioninclude, but are not limited to, sucrose, trehalose, and the like.Furthermore, such carbohydrates and/or sugars may be added to thestorage buffers for the enzymes used in the production of the enzymecompositions and kits of the invention. Such carbohydrates and/or sugarsare commercially available from a number of sources, including Sigma(St. Louis, Mo.).

[0127] It is often preferable to first dissolve the buffer salts,cofactor salts and carbohydrates or sugars at working concentrations inwater and to adjust the pH of the solution prior to addition of theenzymes. In this way, the pH−sensitive enzymes will be less subject toacid- or alkaline-mediated inactivation during formulation of thepresent compositions.

[0128] To formulate the buffered salts solution, a buffer salt which ispreferably a salt of Tris(hydroxymethyl)aminomethane (TRIS®), and mostpreferably the hydrochloride salt thereof, is combined with a sufficientquantity of water to yield a solution having a TRIS® concentration of5-150 millimolar, preferably 10-60 millimolar, and most preferably about20-60 millimolar. To this solution, a salt of magnesium (preferablyeither the chloride or acetate salt thereof) may be added to provide aworking concentration thereof of 1-10 millimolar, preferably 1.5-8.0millimolar, and most preferably about 3-7.5 millimolar. A salt ofpotassium (preferably a chloride or acetate salt of potassium) may alsobe added to the solution, at a working concentration of 10-100millimolar and most preferably about 75 millimolar. A reducing agentsuch as dithiothreitol may be added to the solution, preferably at afinal concentration of about 1-100 mM, more preferably a concentrationof about 5-50 mM or about 7.5-20 mM, and most preferably at aconcentration of about 10 mM. Preferred concentrations of carbohydratesand/or sugars for inclusion in the compositions of the invention rangefrom about 5% (w/v) to about 30% (w/v), about 7.5% (w/v) to about 25%(w/v), about 10% (w/v) to about 25% (w/v), about 10% (w/v) to about 20%(w/v), and preferably about 10% (w/v) to about 15% (w/v). A small amountof a salt of ethylenediaminetetraacetate (EDTA), such as disodium EDTA,may also be added (preferably about 0.1 millimolar), although inclusionof EDTA does not appear to be essential to the function or stability ofthe compositions of the present invention. After addition of all buffersand salts, this buffered salt solution is mixed well until all salts aredissolved, and the pH is adjusted using methods known in the art to a pHvalue of 7.4 to 9.2, preferably 8.0 to 9.0, and most preferably about8.4.

[0129] To these buffered salt solutions, the enzymes (reversetranscriptases and/or DNA polymerases) are added to produce thecompositions of the present invention. M-MLV RTs are preferably added ata working concentration in the solution of about 1,000 to about 50,000units per milliliter, about 2,000 to about 30,000 units per milliliter,about 2,500 to about 25,000 units per milliliter, about 3,000 to about22,500 units per milliliter, about 4,000 to about 20,000 units permilliliter, and most preferably at aworking concentration of about 5,000to about 20,000 units per milliliter. AMV RTs, MAV RTs, RSV RTs and RAVRTs, including those of the invention described above, are preferablyadded at a working concentration in the solution of about 100 to about5000 units per milliliter, about 125 to about 4000 units per milliliter,about 150 to about 3000 units per milliliter, about 200 to about 2500units per milliliter, about 225 to about 2000 units per milliliter, andmost preferably at a working concentration of about 250 to about 1000units per milliliter. The enzymes in the thermophilic DNA polymerasegroup (Taq, Tne, Tma, Pfu, VENT™, DEEPVENT™, Tth and mutants, variantsand derivatives thereof) are preferably added at a working concentrationin the solution of about 100 to about 1000 units per milliliter, about125 to about 750 units per milliliter, about 150 to about 700 units permilliliter, about 200 to about 650 units per milliliter, about 225 toabout 550 units per milliliter, and most preferably at a workingconcentration of about 250 to about 500 units per milliliter. Theenzymes may be added to the solution in any order, or may be addedsimultaneously.

[0130] The compositions of the invention may further comprise one ormore nucleotides, which are preferably deoxynucleoside triphosphates(dNTPs) or dideoxynucleoside triphosphates (ddNTPs). The dNTP componentsof the present compositions serve as the “building blocks” for newlysynthesized nucleic acids, being incorporated therein by the action ofthe polymerases, and the ddNTPs may be used in sequencing methodsaccording to the invention. Examples of nucleotides suitable for use inthe present compositions include, but are not limited to, dUTP, dATP,dTTP, dCTP, dGTP, dITP, 7-deaza-dGTP, α-thio-dATP, α-thio-dTTP,α-thio-dGTP, α-thio-dCTP, ddUTP, ddATP, ddTTP, ddCTP, ddGTP, ddITP,7-deaza-ddGTP, α-thio-ddATP, α-thio-ddTTP, α-thio-ddGTP, α-thio-ddCTP orderivatives thereof, all of which are available commercially fromsources including Life Technologies, Inc. (Rockville, Md.), New EnglandBioLabs (Beverly, Mass.) and Sigma Chemical Company (Saint Louis, Mo.).The nucleotides may be unlabeled, or they may be detectably labeled bycoupling them by methods known in the art with radioisotopes (e.g., ³H,¹⁴C, ³²P or ³⁵S), vitamins (e.g., biotin), fluorescent moieties (e.g.,fluorescein, rhodamine, Texas Red, or phycoerythrin), chemiluminescentlabels (e.g., using the PHOTO-GENE™ or ACES™ chemiluminescence systems,available commercially from Life Technologies, Inc., Rockville, Md.),dioxigenin and the like. Labeled nucleotides may also be obtainedcommercially, for example from Life Technologies, Inc. (Rockville, Md.)or Sigma Chemical Company (Saint Louis, Mo.). In the presentcompositions, the nucleotides are added to give a working concentrationof each nucleotide of about 10-4000 micromolar, about 50-2000micromolar, about 100-1500 micromolar, or about 200-1200 micromolar, andmost preferably a concentration of about 1000 micromolar.

[0131] To reduce component deterioration, storage of the reagentcompositions is preferably at about 4° C. for up to one day, or mostpreferably at −20° C. for up to one year.

[0132] In another aspect, the compositions and reverse transcriptases ofthe invention may be prepared and stored in dry form in the presence ofone or more carbohydrates, sugars, or synthetic polymers. Preferredcarbohydrates, sugars or polymers for the preparation of driedcompositions or reverse transcriptases include, but are not limited to,sucrose, trehalose, and polyvinylpyrrolidone (PVP) or combinationsthereof See, e.g., U.S. Pat. Nos. 5,098,893, 4,891,319, and 5,556,771,the disclosures of which are entirely incorporated herein by reference.Such dried compositions and enzymes may be stored at varioustemperatures for extended times without significant deterioration ofenzymes or components of the compositions of the invention. Preferably,the dried reverse transcriptases or compositions are stored at 4° C. orat −20° C.

[0133] Production of cDNA Molecules

[0134] Sources of Nucleic Acid Molecules

[0135] In accordance with the invention, cDNA molecules (single-strandedor double-stranded) may be prepared from a variety of nucleic acidtemplate molecules. Preferred nucleic acid molecules for use in thepresent invention include single-stranded or double-stranded DNA and RNAmolecules, as well as double-stranded DNA:RNA hybrids. More preferrednucleic acid molecules include messenger RNA (mRNA), transfer RNA (tRNA)and ribosomal RNA (rRNA) molecules, although mRNA molecules are thepreferred template according to the invention.

[0136] The nucleic acid molecules that are used to prepare cDNAmolecules according to the methods of the present invention may beprepared synthetically according to standard organic chemical synthesismethods that will be familiar to one of ordinary skill. More preferably,the nucleic acid molecules may be obtained from natural sources, such asa variety of cells, tissues, organs or organisms. Cells that may be usedas sources of nucleic acid molecules may be prokaryotic (bacterialcells, including but not limited to those of species of the generaEscherichia, Bacillus, Serratia, Salmonella, Staphylococcus,Streptococcus, Clostridium, Chlamydia, Neisseria, Treponema, Mycoplasma,Borrelia, Legionella, Pseudomonas, Mycobacterium, Helicobacter, Erwinia,Agrobacterium, Rhizobium, Xanthomonas and Streptomyces) or eukaryotic(including fungi (especially yeasts), plants, protozoans and otherparasites, and animals including insects (particularly Drosophila spp.cells), nematodes (particularly Caenorhabditis elegans cells), andmammals (particularly human cells)).

[0137] Mammalian somatic cells that may be used as sources of nucleicacids include blood cells (reticulocytes and leukocytes), endothelialcells, epithelial cells, neuronal cells (from the central or peripheralnervous systems), muscle cells (including myocytes and myoblasts fromskeletal, smooth or cardiac muscle), connective tissue cells (includingfibroblasts, adipocytes, chondrocytes, chondroblasts, osteocytes andosteoblasts) and other stromal cells (e.g., macrophages, dendriticcells, Schwann cells). Mammalian germ cells (spermatocytes and oocytes)may also be used as sources of nucleic acids for use in the invention,as may the progenitors, precursors and stem cells that give rise to theabove somatic and germ cells. Also suitable for use as nucleic acidsources are mammalian tissues or organs such as those derived frombrain, kidney, liver, pancreas, blood, bone marrow, muscle, nervous,skin, genitourinary, circulatory, lymphoid, gastrointestinal andconnective tissue sources, as well as those derived from a mammalian(including human) embryo or fetus.

[0138] Any of the above prokaryotic or eukaryotic cells, tissues andorgans may be normal, diseased, transformed, established, progenitors,precursors, fetal or embryonic. Diseased cells may, for example, includethose involved in infectious diseases (caused by bacteria, fungi oryeast, viruses (including AIDS, HIV, HTLV, herpes, hepatitis and thelike) or parasites), in genetic or biochemical pathologies (e.g., cysticfibrosis, hemophilia, Alzheimer's disease, muscular dystrophy ormultiple sclerosis) or in cancerous processes. Transformed orestablished animal cell lines may include, for example, COS cells, CHOcells, VERO cells, BHK cells, HeLa cells, HepG2 cells, K562 cells, 293cells, L929 cells, F9 cells, and the like. Other cells, cell lines,tissues, organs and organisms suitable as sources of nucleic acids foruse in the present invention will be apparent to one of ordinary skillin the art.

[0139] Once the starting cells, tissues, organs or other samples areobtained, nucleic acid molecules (such as mRNA) may be isolatedtherefrom by methods that are well-known in the art (See, e.g.,Maniatis, T., et al., Cell 15:687-701 (1978); Okayama, H., and Berg, P.,Mol. Cell. Biol. 2:161-170 (1982); Gubler, U., and Hoffman, B. J., Gene25:263-269 (1983)). The nucleic acid molecules thus isolated may then beused to prepare cDNA molecules and cDNA libraries in accordance with thepresent invention.

[0140] In the practice of the invention, cDNA molecules or cDNAlibraries are produced by mixing one or more nucleic acid moleculesobtained as described above, which is preferably one or more mRNAmolecules such as a population of mRNA molecules, with two or morepolypeptides having reverse transcriptase activity, or with one or moreof the compositions of the invention or with one or more of the RSV RTsand/or AMV RTs and/or other ASLV RTs of the invention, under conditionsfavoring the reverse transcription of the nucleic acid molecule by theaction of the enzymes or the compositions to form a cDNA molecule(single-stranded or double-stranded). Thus, the method of the inventioncomprises (a) mixing one or more nucleic acid templates (preferably oneor more RNA or mRNA templates, such as a population of mRNA molecules)with one or more reverse transcriptases of the invention and (b)incubating the mixture under conditions sufficient to make one or morenucleic acid molecules complementary to all or a portion of the one ormore templates. Such methods may include the use of one or more DNApolymerases. The invention may be used in conjunction with methods ofcDNA synthesis such as those described in the Examples below, or othersthat are well-known in the art (see, e.g., Gubler, U., and Hoffman, B.J., Gene 25:263-269(1983); Krug, M. S., and Berger, S. L., Meth.Enzymol. 152:316-325 (1987); Sambrook, J., et al., Molecular Cloning: ALaboratory Manual, 2nd ed., Cold Spring Harbor, N.Y.: Cold Spring HarborLaboratory Press, pp. 8.60-8.63 (1989)), to produce cDNA molecules orlibraries.

[0141] The invention is also particularly directed to methods forreverse transcription of a nucleic acid molecule at elevatedtemperatures. As described in more detail in Example 5, retroviral RTsare generally not used at temperatures above 37° C. to 42° C. to copynucleic acid templates such as RNA molecules because of the limitedthermal stability of these mesophilic enzymes. At these temperatures,however, mRNA secondary structure may interfere with reversetranscription (Gerard, G. F., et al., FOCUS 11:60 (1989); Myers, T. W.,and Gelfand, D. H., Biochem. 30:7661 (1991)), and the specificity ofprimer binding may be reduced during gene-specific reverse transcriptionprocesses, such as RT-PCR, causing high background signal (Myers, T. W.,and Gelfand, D. H., Biochem. 30:7661(1991); Freeman, W. N., et al.,BioTechniques 20:782 (1996)). To help overcome these problems, thepresent invention therefore provides methods of RNA reversetranscription at more elevated temperatures, i.e., above 50° C.

[0142] Therefore, the invention is related to methods for reversetranscription of a nucleic acid molecule comprising (a) mixing a nucleicacid template with one or more polypeptides having reverse transcriptaseactivity; and (b) incubating the mixture at a temperature of about 50°C. or greater and under conditions sufficient to make a first nucleicacid molecule complementary to all or a portion of the nucleic acidtemplate. Nucleic acid templates which may be copied according to thesemethods include, but are not limited to, an RNA molecule (e.g., a mRNAmolecule or a polyA+ RNA molecule) and a DNA molecule (e.g., asingle-stranded or double-stranded DNA molecule). According to theinvention, the first nucleic acid molecule produced by these methods maybe a full length cDNA molecule. While any incubation temperature ofabout 50° C. or greater may be used in the present methods, particularlypreferred incubation temperatures include, but are not limited to,temperatures of about 51° C. or greater, about 52° C. or greater, about53° C. or greater, about 54° C. or greater, about 55° C. or greater,about 56° C. or greater, about 57° C. or greater, about 58° C. orgreater, about 59° C. or greater, about 60° C. or greater, about 61° C.or greater, about 62° C. or greater, about 63° C. or greater, about 64°C. or greater, about 65° C. or greater, about 66° C. or greater, about67° C. or greater, about 68° C. or greater, about 69° C. or greater orabout 70° C. or greater. In other such methods, the incubationtemperature may be over a range of incubation temperatures, includingbut not limited to a temperature range of from about 50° C. to about 70°C., about 51° C. to about 70° C., about 52° C. to about 70° C., about53° C. to about 70° C., about 54° C. to about 70° C., about 55° C. toabout 70° C., about 55° C. to about 69° C., about 55° C. to about 68°C., about 55° C. to about 67° C., about 55° C. to about 66° C., about55° C. to about 65° C., about 56° C. to about 65° C., about 56° C. toabout 64° C. or about 56° C. to about 62° C. The invention is alsodirected to such methods which further comprise incubating the firstnucleic acid molecule under conditions sufficient to make a secondnucleic acid molecule complementary to all or portion of the firstnucleic acid molecule. According to the invention, the first and secondnucleic acid molecules produced by these methods may be DNA molecules,and may form a double stranded DNA molecule which may be a full lengthcDNA molecule. As described for the methods above, the one or morepolypeptides having reverse transcriptase activity that are used inthese higher-temperature methods preferably are reduced or substantiallyreduced in RNase H activity, and may be selected from the groupconsisting of one or more AMV reverse transcriptases or subunits thereof(or derivatives, variants, fragments or mutants thereof), and one ormore RSV reverse transcriptases or subunits thereof (or derivatives,variants, fragments or mutants thereof) or other ASLV RTs or subunitsthereof (or derivatives, variants, fragments or mutants thereof).Particularly preferred AMV RTs and RSV RTs and other ASLV RTs includethose provided by the present invention and described in detail above.More particularly preferred are those AMV RTs and RSV RTs having thegenotype AMV αH⁻/H⁺ RT or RSV αH⁻/βH⁺ RT. Such constructs are preferablymade by mutating or modifying the gene encoding the α subunit to reduceor substantially reduce RNase H activity while the gene encoding the βsubunit is not so mutated or modified. The resulting polypeptides(produced by co-expression) will be reduced or substantially reduced inRNase H activity.

[0143] Other methods of cDNA synthesis which may advantageously use thepresent invention will be readily apparent to one of ordinary skill inthe art.

[0144] Having obtained cDNA molecules or libraries according to thepresent methods, these cDNAs may be isolated for further analysis ormanipulation. Detailed methodologies for purification of cDNAs aretaught in the GENETRAPPER™ manual (Life Technologies, Inc.; Rockville,Md.), which is incorporated herein by reference in its entirety,although alternative standard techniques of cDNA isolation such as thosedescribed in the Examples below or others that are known in the art(see, e.g., Sambrook, J., et al., Molecular Cloning: A LaboratoryManual, 2nd ed., Cold Spring Harbor, N.Y.: Cold Spring Harbor LaboratoryPress, pp. 8.60-8.63 (1989)) may also be used.

[0145] In other aspects of the invention, the invention may be used inmethods for amplifying and sequencing nucleic acid molecules. Nucleicacid amplification methods according to this aspect of the invention maybe one- step (e.g., one-step RT-PCR) or two-step (e.g., two-step RT-PCR)reactions. According to the invention, one-step RT-PCR type reactionsmay be accomplished in one tube thereby lowering the possibility ofcontamination. Such one-step reactions comprise (a) mixing a nucleicacid template (e.g., mRNA) with two or more polypeptides having reversetranscriptase activity and with one or more DNA polymerases and (b)incubating the mixture under conditions sufficient to amplify a nucleicacid molecule complementary to all or a portion of the template.Alternatively, amplification may be accomplished by mixing a templatewith two or more polypeptides having reverse transcriptase activity (andoptionally having DNA polymerase activity). Incubating such a reactionmixture under appropriate conditions allows amplification of a nucleicacid molecule complementary to all or a portion of the template. Suchamplification may be accomplished by the reverse transcriptase activityalone or in combination with the DNA polymerase activity. Two-stepRT-PCR reactions may be accomplished in two separate steps. Such amethod comprises (a) mixing a nucleic acid template (e.g., mRNA) withtwo or more reverse transcriptases, (b) incubating the mixture underconditions sufficient to make a nucleic acid molecule (e.g., a DNAmolecule) complementary to all or a portion of the template, (c) mixingthe nucleic acid molecule with one or more DNA polymerases and (d)incubating the mixture of step (c) under conditions sufficient toamplify the nucleic acid molecule. For amplification of long nucleicacid molecules (i.e., greater than about 3-5 Kb in length), acombination of DNA polymerases may be used, such as one DNA polymerasehaving 3′ exonuclease activity and another DNA polymerase beingsubstantially reduced in 3′ exonuclease activity. An alternativetwo-step procedure comprises the use of two or more polypeptides havingreverse transcriptase activity and DNA polymerase activity (e.g., Tth,Tma or Tne DNA polymerases and the like) rather than separate additionof a reverse transcriptase and a DNA polymerase.

[0146] Nucleic acid sequencing methods according to this aspect of theinvention may comprise both cycle sequencing (sequencing in combinationwith amplification) and standard sequencing reactions. The sequencingmethod of the invention thus comprises (a) mixing a nucleic acidmolecule to be sequenced with one or more primers, two or more reversetranscriptases, one or more nucleotides and one or more terminatingagents, (b) incubating the mixture under conditions sufficient tosynthesize a population of nucleic acid molecules complementary to allor a portion of the molecule to be sequenced, and (c) separating thepopulation to determine the nucleotide sequence of all or a portion ofthe molecule to be sequenced. According to the invention, one or moreDNA polymerases (preferably thermostable DNA polymerases) may be used incombination with or separate from the reverse transcriptases.

[0147] Amplification methods which may be used in accordance with thepresent invention include PCR (U.S. Pat. Nos. 4,683,195 and 4,683,202),Strand Displacement Amplification (SDA; U.S. Pat. No. 5,455,166; EP 0684 315), and Nucleic Acid Sequence-Based Amplification (NASBA; U.S.Pat. No. 5,409,818; EP 0 329 822). Nucleic acid sequencing techniqueswhich may employ the present compositions include dideoxy sequencingmethods such as those disclosed in U.S. Pat. Nos. 4,962,022 and5,498,523, as well as more complex PCR-based nucleic acid fingerprintingtechniques such as Random Amplified Polymorphic DNA (RAPD) analysis(Williams, J. G. K., et al., Nucl. Acids Res. 18(22):6531-6535, 1990),Arbitrarily Primed PCR (AP-PCR; Welsh, J., and McClelland, M., Nucl.Acids Res. 18(24):7213-7218, 1990), DNA Amplification Fingerprinting(DAF; Caetano-Anolles et al., Bio/Technology 9:553-557, 1991),microsatellite PCR or Directed Amplification of Minisatellite-region DNA(DAMD; Heath, D. D., et al., Nucl. Acids Res. 21(24): 5782-5785, 1993),and Amplification Fragment Length Polymorphism (AFLP) analysis (EP 0 534858; Vos, P., et al., Nucl Acids Res. 23(21):4407-4414, 1995; Lin, J.J., and Kuo, J., FOCUS 17(2):66-70, 1995). In a particularly preferredaspects, the invention may be used in methods of amplifying orsequencing a nucleic acid molecule comprising one or more polymerasechain reactions (PCRs), such as any of the PCR-based methods describedabove.

[0148] Kits

[0149] In another embodiment, the present invention may be assembledinto kits for use in reverse transcription or amplification of a nucleicacid molecule, or into kits for use in sequencing of a nucleic acidmolecule. Kits according to this aspect of the invention comprise acarrier means, such as a box, carton, tube or the like, having in closeconfinement therein one or more container means, such as vials, tubes,ampules, bottles and the like, wherein a first container means containsone or more polypeptides having reverse transcriptase activity. Thesepolypeptides having reverse transcriptase activity may be in a singlecontainer as mixtures of two or more polypeptides, or in separatecontainers. The kits of the invention may also comprise (in the same orseparate containers) one or more DNA polymerases, a suitable buffer, oneor more nucleotides and/or one or more primers.

[0150] In a specific aspect of the invention, the reverse transcriptionand amplification kits may comprise one or more components (in mixturesor separately) including one or more, preferably two or more,polypeptides having reverse transcriptase activity of the invention, oneor more nucleotides needed for synthesis of a nucleic acid molecule,and/or a primer (e.g., oligo(dT) for reverse transcription). Suchreverse transcription and amplification kits may further comprise one ormore DNA polymerases. Sequencing kits of the invention may comprise oneor more, preferably two or more, polypeptides having reversetranscriptase activity of the invention, and optionally one or more DNApolymerases one or more terminating agents (e.g., dideoxynucleosidetriphosphate molecules) needed for sequencing of a nucleic acidmolecule, one or more nucleotides and/or one or more primers. Preferredpolypeptides having reverse transcriptase activity, DNA polymerases,nucleotides, primers and other components suitable for use in thereverse transcription, amplification and sequencing kits of theinvention include those described above. The kits encompassed by thisaspect of the present invention may further comprise additional reagentsand compounds necessary for carrying out standard nucleic acid reversetranscription, amplification or sequencing protocols. Such polypeptideshaving reverse transcriptase activity of the invention, DNA polymerases,nucleotides, primers, and additional reagents, components or compoundsmay be contained in one or more containers, and may be contained in suchcontainers in a mixture of two or more of the above-noted components ormay be contained in the kits of the invention in separate containers.

[0151] Use of Nucleic Acid Molecules

[0152] The nucleic acid molecules or cDNA libraries prepared by themethods of the present invention may be further characterized, forexample by cloning and sequencing (i.e., determining the nucleotidesequence of the nucleic acid molecule), by the sequencing methods of theinvention or by others that are standard in the art (see, e.g., U.S.Pat. Nos. 4,962,022 and 5,498,523, which are directed to methods of DNAsequencing). Alternatively, these nucleic acid molecules may be used forthe manufacture of various materials in industrial processes, such ashybridization probes by methods that are well-known in the art.Production of hybridization probes from cDNAs will, for example, providethe ability for those in the medical field to examine a patient's cellsor tissues for the presence of a particular genetic marker such as amarker of cancer, of an infectious or genetic disease, or a marker ofembryonic development. Furthermore, such hybridization probes can beused to isolate DNA fragments from genomic DNA or cDNA librariesprepared from a different cell, tissue or organism for furthercharacterization.

[0153] The nucleic acid molecules of the present invention may also beused to prepare compositions for use in recombinant DNA methodologies.Accordingly, the present invention relates to recombinant vectors whichcomprise the cDNA or amplified nucleic acid molecules of the presentinvention, to host cells which are genetically engineered with therecombinant vectors, to methods for the production of a recombinantpolypeptide using these vectors and host cells, and to recombinantpolypeptides produced using these methods.

[0154] Recombinant vectors may be produced according to this aspect ofthe invention by inserting, using methods that are well-known in theart, one or more of the cDNA molecules or amplified nucleic acidmolecules prepared according to the present methods into a vector. Thevector used in this aspect of the invention may be, for example, a phageor a plasmid, and is preferably a plasmid. Preferred are vectorscomprising cis-acting control regions to the nucleic acid encoding thepolypeptide of interest. Appropriate trans-acting factors may besupplied by the host, supplied by a complementing vector or supplied bythe vector itself upon introduction into the host.

[0155] In certain preferred embodiments in this regard, the vectorsprovide for specific expression (and are therefore termed “expressionvectors”), which may be inducible and/or cell type-specific.Particularly preferred among such vectors are those inducible byenvironmental factors that are easy to manipulate, such as temperatureand nutrient additives.

[0156] Expression vectors useful in the present invention includechromosomal-, episomal- and virus-derived vectors, e.g., vectors derivedfrom bacterial plasmids or bacteriophages, and vectors derived fromcombinations thereof, such as cosmids and phagemids, and will preferablyinclude at least one selectable marker such as a tetracycline orampicillin resistance gene for culturing in a bacterial host cell. Priorto insertion into such an expression vector, the cDNA or amplifiednucleic acid molecules of the invention should be operatively linked toan appropriate promoter, such as the phage lambda P_(L) promoter, the Ecoli lac, trp and tac promoters. Other suitable promoters will be knownto the skilled artisan.

[0157] Among vectors preferred for use in the present invention includepQE70, pQE60 and pQE-9, available from Qiagen; pBS vectors, Phagescriptvectors, Bluescript vectors, pNH8A, pNH16a, pNH18A, pNH46A, availablefrom Stratagene; pcDNA3 available from Invitrogen; pGEX, pTrxfus,pTrc99a, pET-5, pET-9, pKK223-3, pKK233-3, pDR540, pRIT5 available fromPharmacia; and pSPORT1, pSPORT2 and pSV·SPORT1, available from LifeTechnologies, Inc. Other suitable vectors will be readily apparent tothe skilled artisan.

[0158] The invention also provides methods of producing a recombinanthost cell comprising the cDNA molecules, amplified nucleic acidmolecules or recombinant vectors of the invention, as well as host cellsproduced by such methods. Representative host cells (prokaryotic oreukaryotic) that may be produced according to the invention include, butare not limited to, bacterial cells, yeast cells, plant cells and animalcells. Preferred bacterial host cells include Escherichia coli cells(most particularly E. coli strains DH10B and Stb12, which are availablecommercially (Life Technologies, Inc; Rockville, Md.)), Bacillussubtilis cells, Bacillus megaterium cells, Streptomyces spp. cells,Erwinia spp. cells, Klebsiella spp. cells and Salmonella typhimuriumcells. Preferred animal host cells include insect cells (mostparticularly Spodoptera frugiperda SJ9 and Sf21 cells and TrichoplusaHigh-Five cells) and mammalian cells (most particularly CHO, COS, VERO,BHK and human cells). Such host cells may be prepared by well-knowntransformation, electroporation or transfection techniques that will befamiliar to one of ordinary skill in the art.

[0159] In addition, the invention provides methods for producing arecombinant polypeptide, and polypeptides produced by these methods.According to this aspect of the invention, a recombinant polypeptide maybe produced by culturing any of the above recombinant host cells underconditions favoring production of a polypeptide therefrom, and isolationof the polypeptide. Methods for culturing recombinant host cells, andfor production and isolation of polypeptides therefrom, are well-knownto one of ordinary skill in the art.

[0160] It will be readily apparent to one of ordinary skill in therelevant arts that other suitable modifications and adaptations to themethods and applications described herein are obvious and may be madewithout departing from the scope of the invention or any embodimentthereof. Having now described the present invention in detail, the samewill be more clearly understood by reference to the following examples,which are included herewith for purposes of illustration only and arenot intended to be limiting of the invention.

Example 1 Cloning and Expression of RSV RNase H⁻ RT

[0161] General Methods

[0162] RSV H⁻ RT is a cloned form of retrovirus RT, in which both the aand the β subunits are mutated by a single amino acid change toeliminate RNase H activity. An RSV RT exhibiting substantially reducedRNase H activity is also produced when only the α subunit is mutated tosubstantially reduce RNase H activity (with the β subunit not beingmutated in the RNase H domain). Mutations and plasmid constructions wereconducted using standard molecular biology methods (see, e.g., Sambrook,J., et al., Molecular Cloning: A Laboratory Manual, 2nd Ed., Cold SpringHarbor, N.Y.: Laboratory Press (1989)), modified as described below.

[0163] Plasmid preparation. Plasmid preparations from 1 ml E. colicultures were made by the alkaline lysis procedure (Sambrook, J., etal., Molecular Cloning: A Laboratory Manual, 2nd ed., Cold SpringHarbor, N.Y.: Laboratory Press (1989)). From 10 ml cultures, thepreparation was additionally treated with phenol-chloroform,precipitated with ethanol, and resuspended in 50 μl of Tris-EDTA (TE)buffer (20 mM Tris-HCl, 1 mM EDTA, pH 8.0). For bacmid preparations,care was taken to avoid shear of the DNA during handling (i.e., novortexing; phenol-chloroform extractions were gently rolled rather thanshaken; slow pipetting).

[0164] PCR. Polymerase chain reactions were carried out in aPerkin-Elmer 9600 thermocycler. Reaction mixtures (50 μl each) contained0.5 units of Taq DNA polymerase, 1 μM of each oligonucleotide, 50 μMeach of dCTP, dGTP, dTTP, and dATP, and about 100 ng of target DNA in areaction buffer consisting of 50 mM KCl, 20 mM Tris-HCl (pH 8.3) and 5mM MgCl₂. Unless otherwise noted, the cycling conditions for each PCRwere 5 minutes at 94° C., followed by eight cycles of 15 seconds at 55°C./30 seconds at 72° C./15 seconds at 94° C., and then 1 minute at 72°C.

[0165] Gel electrophoresis and DNA fragment isolation and cloning. DNAwas electrophoresed at about 10 V/cm in agarose gels inTris-acetate-EDTA buffer (Sambrook, J., et al., Molecular Cloning: ALaboratory Manual, 2nd ed., Cold Spring Harbor, N.Y.: Laboratory Press(1989)) containing about 0.3 μg/ml ethidium bromide. Fragments werevisualized with ultraviolet light and isolated from gel slices by theGlassMAX method (Simms, D., et al., Focus 13:99 (1991)). DNA ligationreactions were performed using T4 DNA ligase under standard conditions(King, P. W., and Blakesley, R. W., Focus 8:1 (1986)), and E. coli DH10Bcells were transformed by a modification of the CaCl₂ method (Lorow, D.,and Jessee, J., Focus 12:19 (1990)).

[0166] Insect cell culture and baculovirus production. Samples (1 ml) ofSf21 insect cells at 5×10⁵ cells/ml were transfected with a mixture of 1μg of bacmid DNA and 8 μl of Cellfectin in 0.2 ml SF900-II serum-freeinsect cell culture medium (Life Technologies, Inc.; Rockville, Md.; seeGodwin, G., and Whitford, W., Focus 15:44 (1993)), according topublished procedures (Anderson, D., et al., Focus 16:53 (1995)). Forgrowth and propagation, Sf9 and Sf21 insect cells were passaged inSF900-II medium at 27° C. in a shaking incubator at 100 rpm (for 600 mlcultures) or 130 rpm (for all other cultures). Care was taken to avoidallowing the cultures to exceed 4×10⁶ cells/ml during growth or to dropbelow 0.5×10⁶ cells/ml during dilution. To expand viral populations,Sf21 cells at about 1×10⁶ cells/ml were infected with enough viral stock(about 0.2% (v/v) virus/culture) to allow growth to about 2×10⁶cells/ml, but not more than 4×10⁶ cells/ml. After 72 hours, the culturewas centrifuged (2,000 rpm for 10 min) and the supernatant was decantedand stored in the dark at 4° C. For infection of cells for proteinproduction, Sf21 insect cells at about 1.5×10⁶ cells/ml were infectedwith enough virus to allow no growth or growth to less than 2.5×10⁶cells/ml. After 72 hours, the culture was harvested by centrifugation at1,000 rpm for 5 minutes and cells were resuspended in 15 ml of PBS (0.2g/liter KCl, 0.2 g/liter KH₂PO₄, 8 g/liter NaCl, 1.15 g/liter Na₂HPO₄,2.16 g/liter Na₂HPO₄.7H₂O) per 500 ml culture.

[0167] Cloning and Expression of Genes Encoding the RSV RT α and βSubunits

[0168] Both the α and β subunits of RSV RT are produced by proteolyticprocessing of larger polypeptide precursors (Gerard, G. F., in: Enzymesof Nucleic Acid Synthesis and Modification, Vol. I: DNA Enzymes, Jacob,S. T., ed., Boca Raton, Fla.: CRC Press, pp. 1-38 (1983)). To obviatethe requirement for proteolytic processing, the coding sequence for RSVRT was mutagenized and subcloned such that both the α and β subunitswere encoded by genes with standard start and stop translationalsignals. Both genes were mutagenized in the RNase H region, althoughconstruction of any combination of subunits (e.g., α RNase H⁻/β RNaseH⁺; α RNase H⁺/β RNase H⁺; α RNase H⁺/β RNase H⁻; α RNase H⁻/β RNase H⁻)may be accomplished in this same manner. It has been discovered that RSVRT α RNase H⁻/β RNase H⁺ is substantially reduced in RNase H activity(approximately 5% of wildtype). A sequence encoding an affinity tag wasadded to the carboxy end of the β subunit.

[0169] Mutagenesis and subcloning of the amino end, the carboxy end andthe middle of the RS VRT β subunit. The RSV RT gene was mutagenized toadd an ATG codon and an NdeI site to the amino end of the sequencecoding for the mature RT polypeptide. This mutagenesis was accomplishedby PCR using a pJD100 target (FIGS. 1, 7) (Wilkerson, V. W., et al., J.Virol. 55:314-321 (1985)) and the following oligonucleotides: (SEQ IDNO:1) AUG GAG AUC UCU CAT ATG ACT GTT GCG CTA CAT CTG GCT (SEQ ID NO:2)AAC GCG UAC UAG U GTT AAC AGC GCG CAA ATC ATG CAG.

[0170] PCR was performed, and PCR products purified, as described aboveand the PCR products were cloned into pAMP18 by UDG cloning (Buchman, G.W., et al., Focus 15:36 (1993)) to form plasmid pAMP18N (FIGS. 1, 8).

[0171] Following mutagenesis and cloning of the amino end, the carboxyend of the gene for the β subunit of RSV was mutagenized and subclonedfrom PJD 100 by PCR and UDG cloning into pAMPI (FIG. 1), using thefollowing oligonucleotides: CUA CUA CUA CUA GGT ACC CTC TCG AAA AGT TAAACC (SEQ ID NO:3) CAU CAU CAU CAU CTC GAG TTA TGC AAA AAG AGG GCT CGCCTC (SEQ ID NO:4) ATC.

[0172] These oligonucleotides were designed to introduce a translationalstop codon in the β gene at the site in which the “p4” region wascleaved from the βp4 polypeptide, and to add an XhoI site after the endof the gene. The PCR products were purified by gel electrophoresis andcloned into pAMP1 by UDG cloning, forming pAMP1C (FIGS. 1, 9). Note thatthis carboxy end is without a His tag, which was added later to form thefinal construct.

[0173] To add the middle region of the RSV RT β subunit, the 2.3 kbHpaI-KpnI fragment from pJD100 (FIG. 1) that encodes the middle of the βsubunit of RSV RT was cloned into the HpaI-KpnI sites of pAMP18N,forming pAMP18NM (FIGS. 1, 10). To add the carboxy end of the RSV RT βsubunit, the 113 bp KpnI-EcoRI fragment encoding the carboxy end of theβ subunit gene was cloned from pAMP1C into the KpnI-EcoRI sites ofpAM18NM, forming pAMP18B (FIGS. 2, 11).

[0174] Following construction of the RSV RT β subunit, which containsRNase H activity, this gene was mutagenized by site-directed mutagenesisto produce a construct encoding a RSV RT β subunit that is substantiallyreduced in RNase H activity (i.e., “RNase H−”). A 3 Kb PstI fragmentfrom pJD100 (FIG. 2) containing the entire RT gene was cloned into M13mp19, forming M13RT (FIGS. 2, 12). Single-stranded DNA containing uracilwas isolated from E. coli strain CJ236 (Bio-Rad; Hercules, Calif.; andCathy Joyce, Yale University, New Haven, Conn.) after infection with M13RT phage containing the PstI fragment. To mutate the RNase H region andto introduce an SstII site, the following oligonucleotide was used: (SEQID NO:5) GGA CCC ACT GTC TTT ACC GCG GCC TCC TCA AGC ACC

[0175] This oligonucleotide induced the substitution of an alanineresidue in place of the aspartate residue at position 450 of the RT,forming M13RTH⁻ (FIGS. 2, 13). In an alternative approach to generate aRNase H⁻ RSV RT, Glu484 may be mutated to glutamine and/or Asp505 may bemutated to asparagine. To convert the β subunit back to RNase H⁺, anoligonucleotide primer having the wildtype sequence may be used.Alternatively, deletions or insertions can be made in the RNase H regionto substantially reduce RNase H activity. DNA sequencing was used toconfirm the Asp450→Ala450 mutation, and the 426 bp BglII-BstEII fragmentfrom M13RTH⁻ was cloned into the BglII-BstEII sites of pAMP18B,replacing the RNase H region and forming pAMP18BH⁻ (FIGS. 2, 14).

[0176] Mutagenesis and subcloning of the gene encoding the RSVRT αsubunit. To create a gene which codes for the a subunit of RSV RT, twooligonucleotides were used to mutagenize the amino end of the RNase H⁻mutant RSV RT gene from pDBH− (FIGS. 3, 15) and to introduce atranslational stop codon where avian retroviral protease p15 normallycleaves the precursor polyprotein to make the α subunit: (SEQ ID NO:6)CAU CAU CAU CAU CCC GGG TTA ATA CGC TTG GAA GGT GGC (SEQ ID NO:7) CUACUA CUA CUA TCA TGA CTG TTG CGC TAC ATC TG

[0177] PCR cycling conditions were 5 minutes at 94° C., followed by 8cycles of 15 seconds at 55° C./2 minutes at 72° C./15 seconds at 94° C.,and then 2 minutes at 72° C. The PCR products were cloned into pAMPI byUDG cloning as described above, forming pAMPIA (FIGS. 3, 16).

[0178] Addition of a His₆ tag to the carboxy end of the RSV β subunit. AHis₆ tag was added to the carboxy end of the RSV RT β subunit bysite-directed mutagenesis, and the mutant sequence was subcloned frompJD100 by PCR and UDG cloning as described above, using the followingoligonucleotides: CUA CUA CUA CUA GGT ACC CTC TCG AAA AGT TAA (SEQ IDNO:8) CAU CAU CAU CAU GAG GAA TTC AGT GAT GGT GAT GGT GAT GTG (SEQ IDNO:9) CAA AAAG AGG

[0179] These oligonucleotides were designed to introduce a translationalstop codon in the gene, and to add a histidine tag to the end of theprotein. The gene product was thus a polypeptide to which 6 histidineswere added to the carboxy end. The PCR products were purified by gelelectrophoresis and inserted into pAMPI by UDG cloning, formingpAMPChis.

[0180] To remove the carboxy end of the RSV RT β gene and replace itwith the carboxy end containing a His₆ tag, the number of KpnI sites inthe baculovirus vector containing the RSV RT P gene had to be reduced.Plasmid pDBH− (FIG. 4) was cleaved with AflIII, and the site was“blunted” with the Klenow fragment of E. coli DNA polymerase I, thepolymerase was inactivated by heat treatment, and the vector was furthercleaved with PvuII (FIG. 4). The deleted vector (5.9 kb) was thenpurified by gel electrophoresis, ligated to itself and used to transformE. coli strain DH10B, forming pDBH−Kpn (FIGS. 4, 17). The KpnI-SstIfragment with the carboxy end of β with the His6 tag was cloned frompAMPChis into the KpnI-SstI sites of pDBH−Kpn, forming pDBH−KpnHis(FIGS. 4, 18).

[0181] Cloning of RSV α and β subunits into a baculovirus expressionvector. During the course of this work, a construct was made in whichthe RSV RT β gene was to be expressed in a baculovirus vector with aHis₆ tag at the amino end. However, a mutation introduced during theconstruction caused the reading frame of the tag to be different fromthat of the β gene. Since portions of this construct were used toconstruct the final baculovirus expression vector, its construction isdescribed here.

[0182] To introduce a His₆ tag and an NdeI site into the baculovirusvector, the following oligonucleotides were annealed and cloned intopFastBac Dual (Harris, R., and Polayes, D. A., Focus 19:6-8 (1997); FIG.5): ACTG GAA TTC ATG CCA ATC CAT CAC CAT CAC CAT CAC CCG T (SEQ IDNO:10) ACGT GTC GAC CAT ATG GAT GAC TAG GTG AAA CGG GTG ATG G (SEQ IDNO:11)

[0183] Both oligonucleotides were formulated into TE buffer at aconcentration of 100 μM, and 5 μl of each oligonucleotide formulationwere mixed with 15 μl of water and 2 μl of 10× React2 buffer (500 mMNaCl, 500 mM Tris-HCl, 100 mM MgCl₂, pH 8.0) in a single tube. This tubewas heated in a 65° C. water bath and cooled slowly over 60 minutes to25° C. The resulting product was a double-stranded DNA molecule with anEcoRI site at the 5′ end (underlined), and a SalI site at the 3′ end(italicized): 5′-ACTG GAA TTC ATG CCA ATC CAT CAC CAT CAC CAT CAC CCG(SEQ ID NO:12) TTT CAC CTA GTC ATC CAT ATG GTC GAC ACGT-3′

[0184] This product was cleaved with EcoRI and SalI and the desiredfragment was cloned into the EcoRI-SalI sites of vector pFastBac Dual bystandard techniques (Harris, R., and Polayes, D. A., Focus 19:6-8(1997)), resulting in the formation of plasmid pFastBac Dual Nde (FIG.5). The NdeI-XhoI B fragment of pAMP18BH— was cloned into the NdeI-SalIsites of pFastBac Dual Nde to create pDBH⁻ (FIGS. 5, 15).

[0185] To clone the RSV RT a gene into a baculovirus vector, the α genewas excised from pAMP1A with SmaI and BspHI and subcloned into theNcoI-SmaI sites of pFastBac Dual, creating pDA (FIGS. 5, 19). Thisplaced the RSV RT α gene downstream from the baculovirus P10 promoter.The RSV α peptide gene and P10 promoter were excised with RsrII andSmaI, and cloned into the RsrII-SmaI sites of pDBH−, forming pDABH−(FIGS. 4, 20).

[0186] To replace the RSV RT β gene in pDABH− with the carboxyHis₆-tagged β gene in pDBH−KpnHis, pDBH−KpnHis was cleaved with NdeI,the site blunt-ended with Klenow fragment, and the β gene with thecarboxy His₆ tag was then released with SstI (FIG. 6). pDABH⁻ wascleaved with EcoRI, the site blunt-ended with Klenow fragment, and thenthe β gene was removed by digesting with SstI. The vector fragment,which was a blunt-end SstI fragment of pFastBac Dual with the α genecloned in front of the p10 promoter, was ligated to the blunt-end SstIfragment with the carboxy His-tagged β gene from pDBH−KpnHis. E coliDH10B cells were transformed with this construct, and a transformantcontaining pDABH−His (FIGS. 6, 21) was selected.

[0187] The recombinant host cell comprising plasmid pDABH−His, E. coliDH10B(pDABH−His), was deposited on Apr. 15, 1997, with the Collection,Agricultural Research Culture Collection (NRRL), 1815 North UniversityStreet, Peoria, Ill. 61604 USA, as Deposit No. NRRL B-21679.

[0188] Using the deposited plasmid, one of ordinary skill in the art mayeasily produce, using standard genetic engineering techniques (such asthose for site-directed mutagenesis described above), plasmids encodingvarious forms of the α and/or β subunits of RSV RT (e.g., α RNase H⁺/βRNase H⁺; α RNase H⁻/β RNase H⁻; α RNase H⁺/β RNase H⁺; and α RNase H⁻/βRNase H⁺).

[0189] Transfection of insect cellsfor virus and RSVRTproduction. Toprepare vectors for the transfection of insect cells, it was firstnecessary to insert the RSV RT gene constructs into a baculovirusgenome. One method of accomplishing this insertion is by using thesite-specific transposon Tn7 (Luckow, V.A., in: Recombinant DNATechnology andApplications, Prokop, A., et al., eds., New York:McGraw-Hill (1991)). In DH10Bac host cells, most of the baculovirusgenome is represented on a low copy plasmid (a “bacmid”) which alsocontains a Tn7 insertion site within a gene (Harris, R., and Polayes, D.A., Focus 19:6-8 (1997)). Transposition of Tn7 is facilitated by the Tn7transposase, which is produced from a second plasmid in DH10Bac hostcells, and pFastBac DUAL is constructed with the “right” and “left”regions of Tn7 flanking the promoter and cloning sites.

[0190] To insert the present constructs into a bacmid expression vector,pDABH−His was transformed into DH10Bac cells and transposition ofsequences encoding the RSV RT β gene (whose synthesis is directed by thebaculovirus polyhedrin promoter) and the RSV RT α gene (whose synthesisis directed by the baculovirus P10 promoter) was followed by screeningfor loss of β-galactosidase activity following transposition to the siteon the bacmid which codes for the β-galactosidase a peptide (Harris, R.,and Polayes, D. A., Focus 19:6-8 (1997)). Bacmid DNA was then preparedfrom 10 ml transformant cultures by a slight modification of a standardminiprep procedure (Sambrook, J., et al., Molecular Cloning: ALaboratory Manual, 2nd Ed., Cold Spring Harbor, N.Y.: Cold Spring HarborLaboratory Press (1989)). About 1 μg of the bacmid DNA was then used totransfect Sf21 insect cells using the cationic lipid Cellfectin(Anderson, D., et al., Focus 16:53 (1995)).

[0191] For expansion of primary virus, the supernatant was removed fromthe transfected cells about 72 hours after transfection and 1 ml wasused to infect 35 ml of Sf21 insect cells (about 1.2×10⁵ cells/ml).After 72 hours, the culture was centrifuged (2,000 rpm; 10 min) and thesupernatant was decanted and used as a secondary viral stock. Thesecondary virus stock was expanded similarly by infecting 35 ml of Sf21cells with 0.1 ml of the secondary stock.

[0192] Virus stocks were then used to infect Sf2 1 cells for theexpression of RSV RT. In preliminary experiments, expression of RTactivity from infected cells was found to be maximal about 72 hoursafter infection. For test expressions, 70 ml of Sf21 cells were infectedwith 5 ml of the virus stock, and the cells were harvested 72 hoursafter infection by centrifugation at 1,000 rpm for 5 minutes,resuspension in PBS (2.5% of the culture volume) and recentrifugation at1,000 rpm for 5 minutes. Supernatants were removed and the cells werestored at −70° C. until use. For larger scale production, 600 ml ofcells in 2.8 liter Fernbach flasks were infected with 2 ml of virusstock, and the cells were harvested 72 hours after infection.

Example 2 Isolation of RSV RNase H⁻ RT

[0193] To provide purified recombinant RSV H⁻ RT, cloned RSV H⁻ RT wasoverexpressed in cultured insect cells as described in Example I andpurified by affinity and ion-exchange chromatography. The RSV RTproduced comprises the α and β subunits. Isolation of RSV RT provides asubstantially pure RSV RT in which contaminating enzymes and otherproteins have been substantially removed, although such contaminantsneed not be completely removed.

[0194] Buffers. The pH of all buffers was determined at 23° C., andbuffers were stored at 4° C. until use. Crack Buffer contained 50 mMTris-HCl (pH 7.9), 0.5 M KCl, 0.02% (v/v) Triton X-100 and 20% (v/v)glycerol. Just before use, the following protease inhibitors (BoehringerMannheim; Indianapolis, Ind.) were added to Crack Buffer at the finalconcentrations indicated: leupeptin (2 μg/ml), Pefabloc (48 μg/ml),pepstatin A (2 μg/ml), benzamidine (800 μg/ml) and PMSF (50 μg/ml).Buffer A contained 20 mM Tris-HCl (pH 7.9), 0.25 M KCl, 0.02% (v/v)Triton X-100, and 10% (v/v) glycerol. Buffer B was Buffer A with 1 Mimidazole added. Buffer S contained 50 mM Tris-HCl (pH 8.2), 0.02% (v/v)Triton X-100, 10% (v/v) glycerol, 0.1 mM EDTA and 1 mM dithiothreitol(DTT). Buffer T was Buffer S with 1 M KCl added. Buffer H contained 20mM potassium phosphate (pH 7.1), 0.02% (v/v) Triton X-100, 20% (v/v)glycerol, 0.1 mM EDTA and 1 mM DTT. Buffer J was Buffer H with 1 M KCladded. Storage Buffer contained 200 mM potassium phosphate (pH 7.1),0.05% (v/v) NP-40, 50% glycerol (v/v), 0.1 mM EDTA, 1 mM DTT, and 10%(w/v) trehalose.

[0195] Extract preparation. Frozen insect cells (25 g) were thawed and aslurry prepared at 4° C. with 50 nml of Crack Buffer plus inhibitors.Cells were disrupted at 4° C. by sonication with a Fisher 550 Sonicatorat 25% of maximum power. The disrupted crude extract was clarified bycentrifugation at 27,000×g for 30 minutes at 4° C.

[0196] RSVR Tisolation. Following clarification, the extract wasfractionated and RSV RT purified by column chromatography at 4° C. Theclarified crude extract was loaded unto a 30 ml Chelating Sepharose FastFlow column (Pharmacia; Piscataway, N.J.) charged with NiSO₄ as permanufacturer's instructions and equilibrated in 99.5% Buffer A+0.5%Buffer B. The column was washed with one column volume of 99% BufferA+1% Buffer B and then with 10 column volumes of 98.5% Buffer A+1.5%Buffer B. RT was eluted with a 10-column volume linear gradient of 98.5%Buffer A+1.5% Buffer B to 75% Buffer A+25% Buffer B.

[0197] During purification, reverse transcriptase activity was assayedwith poly(C)-oligo(dG), which is specific for reverse transcriptase(Gerard G. F., et al, Biochemistry 13:1632-1641 (1974)). RT unitactivity was defined and assayed as described (Houts, G. E., et al., J.Virol. 29:517-522 (1979)). Using the poly(C)-oligo(dG) assay, the peakfractions of RT activity from the Chelating Sepharose Fast Flow column(10 to 17% Buffer B) were pooled, diluted with an equal volume of BufferS, and loaded on a 5 ml AF-Heparin-650M column (TosoHaas;Montgomeryville, Pa.) equilibrated in Buffer S. After a wash with 12column volumes of 90% Buffer S+10% Buffer T, the column was eluted witha 15-column volume linear gradient of Buffer S to 30% Buffer S+70%Buffer T. The peak fractions of RT activity (43 to 50% Buffer T) werepooled, diluted with 2.5 volumes of Buffer H, and loaded unto a Mono SHR 5/5 column (Pharmacia; Piscataway, N.J.) equilibrated in Buffer H.After a wash with 20 column volumes of 85% Buffer H+15% Buffer J, thecolumn was eluted with a 20 column volume gradient of 85% Buffer H+15%Buffer J to 50% Buffer H+50% Buffer J. The RT peak fractions werepooled, dialyzed against Storage Buffer overnight, and stored at −20° C.

[0198] Following purification, RSV H⁻ RT was found to be >95%homogeneous as judged by SDS-PAGE. The purified enzyme was also found tobe substantially lacking in RNase and DNase contamination, andsubstantially reduced in RNase H activity.

Example 3 Preparation of Full-Length cDNA Molecules

[0199] Enzymes. SuperScript II RT (SS II RT), a cloned form of Moloneymurine leukemia virus (M-MLV) RT lacking demonstrable RNase H activity(i.e., an “RNase H⁻ RT”), was from Life Technologies, Inc. (Rockville,Md.). M-MLV RT, a cloned murine RT with full RNase H activity (i.e., an“RNase H⁺ RT”), was also from Life Technologies, Inc. AMV RT, an RNaseH⁺ uncloned form of avian myeloblastosis virus RT, was from SeikagakuAmerica, Inc. RSV H⁻ RT was prepared as described in Examples 1 and 2.Recombinant Tth DNA polymerase, a cloned, thermophilic DNA polymerasefrom Thermus thermophilus with reverse transcriptase activity, was fromPerkin Elmer.

[0200] Synthetic mRNA. A 7.5 kilobase (Kb) synthetic mRNA with a120-nucleotide 3′ poly(A) tail (Life Technologies, Inc.; Rockville, Md.)was used as template to test the efficiency of various enzymes alone orin combination.

[0201] cDNA Synthesis Reaction Mixtures. Reaction mixtures (20 μl each)contained the following components unless specified otherwise: 50 mMTris-HCl (pH 8.4 at 24° C.), 75 mM KCl, 10 mM dithiothreitol, 1 mM eachof [³²P]dCTP (300 cpm/pmole), dGTP, dTTP, and dATP, 25 μg/ml(p(dT)₂₅₋₃₀), 125 μg/ml of 7.5 Kb mRNA, and 35 units of cloned rat RNaseinhibitor. Reaction mixtures with RTs alone or in combination containedthe following: SS II RT alone: 0.5 mM dNTPs, 3 mM MgCl₂, and 200 unitsof SS II RT RSV H⁻ RT alone: 7.5 mM MgCl₂ and 7 units of RSV H⁻ RT; AMVRT alone: 50 mM KCl, 10 mM MgCl₂, 4 mM sodium pyrophosphate and 14 unitsof AMV RT SS II RT plus RSV H⁻ RT: 5 mM MgCl₂, 200 units of SS II RT,and 7 units of RSV H⁻ RT; Tth DNA Polymerase 0.5 mM dNTPs, 1 mM MnCl₂and 5 units of alone: Tth DNA Polymerase M-MLV H⁺ RT alone: 0.5 mMdNTPs, 3 mM MgCl₂, 50 μg/ml actinomycin D and 200 units of M-MLV H⁺ RTTth DNA Polymerase plus same as Tth DNA Polymerase alone, plus either SSII RT or RSV H⁻ either 200 units of SS II RT or 7 units of RT: RSV H⁻ RTM-MLV H⁺ RT plus SS II same as M-MLV H⁺ RT alone, plus 200 RT: units ofSS II RT; M-MLV H⁺ plus either 5 mM MgCl₂, 50 μg/ml actinomycin D, 200AMV RT or RSV H⁻ RT: units of M-MLV H⁺ RT, and either 14 units of AMV RTor 7 units of RSV H⁻ RT AMV RT plus either SS II 5 mM MgCl₂, 50 μg/mlactinomycin D, 14 RT or RSV H⁻ RT: units of AMV RT, and either 200 unitsof SS II RT or 7 units of RSV H⁻ RT

[0202] When RTs were used in combination, one enzyme was added firstfollowed immediately by the addition of an aliquot of the second enzyme.In some cases in which a single enzyme was used, a second aliquot of thesame enzyme was added as a control to assess the effect of doubling theamount of the single enzyme.

[0203] All cDNA synthesis reactions were carried out at 45° C. for 50minutes, and the resultant cDNA product was detectably labeled by theRT-catalyzed incorporation of a ³²P-labeled deoxyribonucleosidetriphosphate precursor. The total yield of cDNA was determined by acidprecipitation of a portion of the cDNA product and counting it in ascintillation counter. The ³²P-labeled cDNA product in the remainder ofthe reaction mixture was fractionated by alkaline agarose gelelectrophoresis (Carmichael, G. G., and McMaster, G. K., Meth. Enzymol.65:380-385 (1980)). The gel was dried and the size distribution of thecDNA product was established by autoradiography. Using theautoradiographic film as a template, the dried gel was cut and analyzedby scintillation counting to establish the fraction of full length (7.5Kb) product synthesized.

[0204] Tables 1 and 2 show the total amount of cDNA synthesized and fulllength cDNA synthesized, respectively, from the 7.5 Kb mRNA by enzymesalone or in combination. The following conclusions can be drawn:

[0205] 1. When RTs were present alone, the highest yields of total andfull length product were obtained with the RNase H⁻ forms of RT. Witheither RSV H⁻ RT or SS II RT, the total yield was almost double thehighest yield obtained with an RNase H⁺ enzyme (1011 and 946 ng,respectively, versus 607 ng). The effect of removing RNase H from thereaction was even more dramatic when full length yields were examined.In this case, yields were at least tripled (234 and 208 ng for RSV H⁻and SS II RT versus 79 and 26 for M-MLV H⁺ RT and AMV RT, respectively).These results demonstrate the dramatic positive effect of eliminatingRNase H from RT.

[0206] 2. When RTs were combined, several effects were observed. MixingRTs from different sources, whether RNase H⁻ or RNase H⁺, increasedtotal and full length yields. This is consistent with the hypothesisthat pausing at sites unique to one enzyme can be reduced by a second RTwith a different set of pause sites. However, by far the greatest yieldsof total and full length cDNA product were obtained when two differentRNase H⁻ RTs were combined (see shaded boxes in Tables 1 and 2). Theseresults indicate that the two RNase H⁻ enzymes cooperate to synthesizefull-length cDNA molecules: the first enzyme synthesizes truncated cDNAmolecules, which are then extended to full-length via the activity ofthe second enzyme. Thus, the compositions and methods of the presentinvention facilitate the synthesis of full-length cDNA molecules. TABLE1 Total Yield of cDNA Synthesized by Various Enzymes from 7.5-Kb mRNA¹

[0207] TABLE 2 Yield of Full Length cDNA Synthesized by Various Enzymesfrom 7.5 Kb mRNA¹

[0208] In the mixing experiments summarized in Tables 1 and 2, tworeverse transcriptases were added simultaneously to a reaction underconditions that may have been suboptimal for a single given RT. This wasthe case when an avian and a murine RT were used together, since theMgCl₂ concentration was set at 5 mM, between the optima of 3 mM and 7.5mM for murine and avian RT, respectively. In addition, full advantagecould not be taken in these experiments of the thermal stability ofavian RNase H⁻ RT in reactions containing a less thermostable murine RT.

[0209] To use multiple enzymes and to address the fact that differentenzymes may have different optimal conditions, sequential additions orseparate use of the enzyme may be done in accordance with the methods ofthe invention. For example, cDNA could be synthesized from differentaliquots of the same RNA in separate reaction tubes with different RTsunder reaction conditions optimal for each RT. Subsequently, the cDNAsfrom each reaction could be mixed before performing furthermanipulations. Alternatively, RTs could be used singly and sequentiallyin one tube to perform cDNA synthesis. That is, SuperScript II could beused first to copy an RNA population under optimal reaction conditions,and then conditions could be adjusted to optimal for RSV H⁻ RT in thesame tube, and further synthesis could be performed with the avian RT atelevated temperature.

Example 4 Cloning and Expression ofAvian Myeloblastosis Virus (AMV) RTand AMV RNase H⁻ (AMV H⁻) RT

[0210] General Methods

[0211] The AMV RT of the present invention is a cloned form of avianretrovirus RT. The AMV H⁻RT is a variant of cloned AMV RT in which boththe α and the β subunits are mutated by a single amino acid change toeliminate RNase H activity, although AMV RT substantially reduced inRNase H activity is also produced by mutating the α subunit alone (the βsubunit not containing a mutation in the RNase H domain). Mutations andplasmid constructions were conducted using standard molecular biologymethods (see, e.g., Sambrook, J., et al., Molecular Cloning: ALaboratory Manual, 2nd Ed., Cold Spring Harbor, N.Y.: Laboratory Press(1989)), modified as described below. Plasmid preparation, PCR, gelelectrophoresis, DNA fragment isolation and cloning, insect cell cultureand baculovirus production were all performed as described for RSV RTcloning and expression in Example 1.

[0212] Cloning and Expression of Genes Encoding the AMV RT α and βSubunits

[0213] To clone AMV RT, AMV viral RNA was prepared (Strauss, E. M., etal., J. Virol. Meth. 1:213 (1980)) from purified (Grandgenett, D. P., etal., Appl. Microbiol. 26:452 (1973)) AMV obtained from Life Sciences(St. Petersburg, Fla.). AMV RT cDNA was prepared from AMV viral RNA withthe SuperScript Plasmid System for cDNA Synthesis and Plasmid Cloning(Life Technologies, Inc.; Rockville, Md.) following the instructions inthe kit manual. A primer specific for AMV RT was used that adds a NotIsite to the cDNA. Following preparation, AMV RT cDNA was cloned intopSPORT1 that had been treated with SalI and NotI, resulting in a vector(pSPORT8) comprising the AMV RT gene (FIG. 22).

[0214] Both the α and β subunits of AMV RT are produced by proteolyticprocessing of larger polypeptide precursors (Gerard, G.F., in: Enzymesof Nucleic Acid Synthesis and Modification, Vol. I: DNA Enzymes, Jacob,S.T., ed., Boca Raton, Fla.: CRC Press, pp. 1-38 (1983)). To obviate therequirement for proteolytic processing, the coding sequence for AMV RTwas mutagenized and subcloned such that both the α and β subunits wereencoded by genes with standard start and stop translational signals. Tomake RNase H⁻ constructs, both α and β genes were mutagenized in theRNase H region, although construction of any combination of subunits(e.g., α RNase H⁻/β RNase H⁺; α RNase H⁺/β RNase H⁺; α RNase H⁺/β RNaseH⁻; α RNase H⁻/β RNase H⁻) may be accomplished in this same manner. Ithas been discovered that AMV RT a RNase H⁻/β RNase H⁺ is substantiallyreduced in RNase H activity (approximately 5% of wildtype). A sequenceencoding an affinity tag was added to the carboxy end of the β subunit.

[0215] Synthesis of cDNA from AMVRNA. The AMV RNA was copied into DNAusing a 3′ primer which is complementary to the 3′ end of the AMV RTgene, and which adds aNotI site to the 3′ end of the gene (FIG. 22). The5′ end of the resulting cDNA was made into a SalI end by the addition ofa SalI adapter. This cDNA was then cloned into a SalI-NotI cleavedvector (pSPORT1). First strand cDNA was synthesized using Superscript IIRT (Gerard, G. F., et al., FOCUS 11:66 (1989)) and a gene specificprimer (Oligonucleotide #1) instead of the NotI primer-adapter:Oligonucleotide #1: 5′ GACTAGTTCTAGATCGCGAGCGGCCGCCCATTAACTCTCGTTGG (SEQID NO:13) CAGC 3′

[0216] The second strand synthesis was achieved using DNA polymerase Iin combination with E. coli RNase H and DNA ligase at 16° C. andsubsequently polishing the termini with T4 DNA polymerase. The cDNA wasdeproteinized and precipitated with ethanol, and SalI adaptersconsisting of Oligonucleotides #2 and #3 were ligated to the cDNA:Oligonucleotide #2: 5′ TCGACCCACGCGTCCG 3′ (SEQ ID NO:14)Oligonucleotide #3: 5′ CGGACGCGTGGG 3′ (SEQ ID NO:15)

[0217] The addition of the adapters was followed by digestion with NotI.Size fractionation of the cDNA was done on 1 ml prepacked columnsprovided with the cDNA cloning kit. The amount of cDNA in each fractionwas calculated from the specific activity of incorporated ³²P label, andthe size of the cDNA was determined by autoradiography of an agarosegel. Those fractions that were greater than 3 Kb were selected forcloning.

[0218] Cloning AMV cDNA into a Vector. The cDNA was ligated intoSalI-NotI cleaved pSPORT1 vector and then the ligated cDNA was used totransform E. coli MAX EFFICIENCY DH10B™ competent cells (LifeTechnologies, Inc., Rockville, Md.). After transformation, aliquots ofcells were plated on LB plates containing ampicillin. Twelve colonieswere picked and 1 ml cultures were grown for mini-preps. Gels were runto check for certain fragments after digesting with restriction enzymesSalI, Mlul, PstI, ApaI, DraIII, SphI and BglII. One plasmid, pSPORT8,was selected since the insert was large enough to code for the AMV RTgene (3 Kb) and a PstI site was present which indicated that the 5′terminus of the AMV RT gene was present (FIG. 22).

[0219] Mutagenesis and subcloning of the amino end, the carboxy end andthe middle of the AMV RT β subunit The AMV RT gene was mutagenized toadd an ATG codon and an EcoRI site to the amino end of the sequencecoding for the mature RT polypeptide by PCR with pSPORT8 as the targetand the following oligonucleotides: Oligonucleotide #4: 5′ AUG GAG AUCUCU GAA TTC ATG ACT GTT GCG CTA CAT CTG (SEQ ID NO:16) GCT 3′Oligonucleotide #5: 5′ AAC GCG UAC UAG U GTT AAC AGC GCG CAA ATC ATG CAG3′ (SEQ ID NO:2)

[0220] PCR was performed, and PCR products purified, as described above.The PCR reaction was treated with DpnI to destroy the target and the PCRproduct was cloned into pAMP18 by UDG cloning (Buchman, G.W., et al.,Focus 15:36 (1993)), forming plasmid pAMVN (FIGS. 23, 26).

[0221] Following mutagenesis and cloning of the amino end, a His₆affinity tag, a XhoI site and a translational stop codon were added tothe carboxy end of the gene for the β subunit of AMV RT in pSPORT8 byPCR using the following oligonucleotides: Oligonucleotide #6: 5′ CUA CUACUA CUA GGT ACC CTC TCG AAA AGT TAA ACC 3′ (SEQ ID NO:3) Oligonucleotide#7: 5′ CAU CAU CAU CAU GAG GAA TTC AGT GAT GGT GAT GGT GAT GTG CAA A AAGAGG 3′ (SEQ ID NO:9)

[0222] PCR was performed, and PCR products purified, as described above.The PCR reaction was treated with DpnI to destroy the target and the PCRproduct was cloned into pAMP18 by UDG cloning (Buchman, G. W., et al.,Focus 15:36 (1993), forming plasmid pAMVC (FIGS. 23, 27).

[0223] To add the middle region of the AMV RT β subunit, the 2.3 KbHpaI-KpnI fragment from pSPORT8 that encodes the middle of the β subunitof AMV RT was cloned into the HpaI-KpnI sites of pAMVN, forming pAMVNM(FIGS. 23, 28). To add the carboxy end of the AMV RT β subunit, sincepAMVC has two KpnI sites (FIG. 27), it was partially cleaved with KpnI,then completely cleaved with ScaI, and the 3 Kb fragment with thecarboxy end of AMV RT was isolated and ligated to the 3.5 Kb ScaI-KpnIfragment of pAMVNMH− (FIG. 29), forming pAMVBH− (FIGS. 23, 30).

[0224] Mutagenesis of the beta subunit to RNase H⁻. The RSV RT and AMVRT genes are related (GenBank sequences J02342, J02021 and J02343 forRSV-C; L10922, L10923, L10924 for AMV). These genes code for anidentical sequence of amino acids over a short distance of the RNase Hregion. As described above in Example 1, during the cloning andmutagenesis of the RSV RT genes an RNase H⁻ derivative of the RSV RT Pgene was made by site-directed mutagenesis. The oligonucleotide that wasused (oligonucleotide #8) changed amino acid Asp450 to an Ala450 andintroduced an SstII site (underlined). Oligonucleotide #8 GGA CCC ACTGTC TTT ACC GCG GCC TCC TCA AGC ACC

[0225] The plasmid with the RNase H− mutation in the RSV RT P gene ispAMP 18BH− (FIGS. 2, 14). The 129 bp BsrGI-BstEII fragment frompAMP18BH− was cloned into the BsrGI-BstEII sites of pAMVNM, replacingthe RNase H⁺ region in this plasmid and forming pAMNVMH⁻ (FIGS. 23, 29).The effect of this replacement was to change Asp450 to Ala450 in the AMVβ gene without changing any other amino acids. To convert the β subunitback to RNase H+, an oligonucleotide primer having the wildtype sequencemay be used.

[0226] Mutagenesis and subcloning of the gene encoding the AMV RT αsubunit. To create a gene which codes for the α subunit of AMV RT,oligonucleotides #9 and #10 were used to mutagenize the amino end of theAMV RT gene from pAMVNM to introduce a translational stop codon whereavian retroviral protease p15 normally cleaves the precursor polyproteinto make the α subunit: Oligonucleotide #9: 5′ CAU CAU CAU CAU CCC GGGTTA ATA CGC TTG GAA GGT GGC 3′ (SEQ ID NO:6) Oligonucleotide #10: 5′ CUACUA CUA CUA TCA TGA CTG TTG CGC TAC ATC TG 3′ (SEQ ID NO:7)

[0227] PCR cycling conditions were 5 minutes at 94° C., followed by 8cycles of 15 seconds at 55° C./2 minutes at 72° C./15 seconds at 94° C.,and then 2 minutes at 72° C. The PCR reaction was treated with DpnI todestroy the target and the PCR product was cloned into pAMP18 by UDGcloning (Buchman, G. W., et al., Focus 15:36 (1993)), forming plasmidpAMVA (FIGS. 24, 31). To make the Rnase H⁻ allele of the a subunit ofAMV RT, the same procedure was followed using pAMVBH− as a target,forming plasmid pAMVAH− (FIGS. 24, 32).

[0228] Cloning the AMV RT α and β genes into pFastBac Dual. The α genewas excised from pAMVA with SmaI and BspHI, and subcloned into theNcoI-PvuII sites of pFastBac Dual (pD; FIG. 33), creating plasmid pDAMVA(FIGS. 25, 34). An RNase H⁻ AMV RT α gene was similarly cloned frompAMVAH−, forming plasmid pDAMVAH− (FIGS. 25, 35). In both plasmids, theAMV RT α gene was downstream from the baculovirus P10 promoter. TheRNase H− AMV RT P gene was excised from pAMVBH− with EcoRI and clonedinto the EcoRI site of pDAMVA. Clones were selected in which the EcoRIinsert was oriented such that the AMV RT β gene was downstream from thepolyhedrin promoter, forming plasmid PDAMVABH− (FIGS. 25, 36). In thisconstruct, the AMV RT α gene was RNase H⁺, but the β gene was RNase H⁻.The AMV RNase H⁻ RT β gene and polyhedrin promoter were excised frompDAMVABH− with RsrII and NotI and cloned into the RsrII-NotI sites ofpSPORT1, forming plasmid pJAMVBH− (FIGS. 25, 37). The AMV RNase H⁻ RT βgene and polyhedrin promoter were excised from pJAMVBH− with RsrII andNotI and cloned into the RsrII-NotI sites of pDAMVAH−, forming plasmidpDAMVAH−BH− (FIGS. 25, 38).

[0229] The recombinant host cell comprising plasmid pDAMVABH−, E. coliDH10B(pDAMVABH−), was deposited on Jun. 17, 1997, with the Collection,Agricultural Research Culture Collection (NRRL), 1815 North UniversityStreet, Peoria, Ill. 61604 USA, as Deposit No. NRRL B-21790.

[0230] Using the deposited plasmid, one of ordinary skill in the art mayeasily produce, using standard genetic engineering techniques (such asthose for site-directed mutagenesis described above), plasmids encodingvarious forms of the α and/or β subunits of AMV RT (e.g., α RNase H⁺/βRNase H⁺; α RNase H⁻/β RNase H⁻; α RNase H⁺/β RNase H⁻; and α RNase H⁻/βRNase H⁺).

[0231] Transfection of insect cells for virus and AMV RT production. Toprepare vectors for the transfection of insect cells, it was firstnecessary to insert the AMV RT gene constructs into a baculovirusgenome. This insertion was accomplished using the site-specifictransposon Tn7, as described for the insertion of the RSV RT geneconstructs into bacmids in Example 1. Plasmid pDAMVABH− was transformedinto DH10Bac cells and transposition of sequences encoding the AMV RT βgene (whose synthesis is directed by the baculovirus polyhedrinpromoter) and the AMV RT α gene (whose synthesis is directed by thebaculovirus P10 promoter) was followed by screening for loss ofβ-galactosidase activity following transposition to the site on thebacmid which codes for the β-galactosidase a peptide (Harris, R., andPolayes, D. A., FOCUS 19:6-8 (1997)). Bacmid DNA was then prepared fromtransformants (10 ml cultures) by a slight modification of a standardminiprep procedure, as described in Example 1. About 1 μg of the bacmidDNA was used to transfect Sf21 insect cells using the cationic lipidCelifectin (Anderson, D., et al., FOCUS 16:53 (1995)).

[0232] For expansion of primary virus, the supernatant was removed fromthe transfected cells about 72 hours after transfection and 1 ml wasused to infect 35 ml of Sf21 insect cells (about 1.2×10⁵ cells/ml).After 72 hours, the culture was centrifuged (2,000 rpm; 10 min) and thesupernatant was decanted and used as a secondary viral stock. Thesecondary virus stock was expanded similarly by infecting 35 ml of Sf21cells with 0.1 ml of the secondary stock.

[0233] Virus stocks were then used to infect Sf21 cells for theexpression of AMV RT. In preliminary experiments, expression of RTactivity by infected cells was found to be maximal about 72 hours afterinfection. For test expressions, 70 ml of Sf21 cells were infected with5 ml of the viral stock, and the cells were harvested 72 hours afterinfection by centrifugation at 1,000 rpm for 5 minutes, resuspension inPBS (2.5% of the culture volume) and recentrifugation at 1,000 rpm for 5min. Supernatants were removed and the cells were stored at −70° C.until used. For larger scale production, 600 ml of cells in 2.8 literFernbach flasks were infected with 2 ml of viral stock, and the cellswere harvested 72 hours after infection. AMV RT was then isolated asdescribed for RSV RT in Example 2.

Example 5 Reverse Transcription with Retroviral RTs at TemperaturesAbove 55° C.

[0234] Retroviral reverse transcriptases have historically been used tocatalyze reverse transcription of mRNA at temperatures in the range of37° C. to 42° C. (see technical literature of commercial suppliers ofRTs such as LTI, Pharmacia, Perkin Elmer, Boehringer Mannheim andAmersham). There is a prevailing belief that at these temperatures mRNAsecondary structure interferes with reverse transcription (Gerard, G.F., et al., FOCUS 11:60 (1989); Myers, T. W., and Gelfand, D. H.,Biochem. 30:7661 (1991)) and the specificity of primer binding isreduced during gene-specific reverse transcription processes, such asRT-PCR, causing high background signal (Myers, T. W., and Gelfand, D.H., Biochem. 30:7661 (1991); Freeman, W. N., et al., BioTechniques20:782 (1996)). It is therefore desirable to carry out RNA reversetranscription at more elevated temperatures, i.e., above 55° C., to helpalleviate these problems.

[0235] As noted above, retroviral RTs are generally not used attemperatures above 37° C. to 42° C. to copy RNA because of the limitedthermal stability of these mesophilic enzymes. In recent years, however,it has been reported that AMV RT can be used to perform RT-PCR of smallamplicons (<500 bases) at 50° C., and to a limited extent at 55° C.(Freeman, W. M., et al., BioTechniques 20:782 (1996); Mallers, F., etal., BioTechniques 18:678 (1995); Wang, R. F., et al., BioTechniques12:702 (1992)). Forms of M-MLV RT lacking RNase H activity, because ofremoval of the RNase H domain (Gerard, G. F., et al., FOCUS 11:66 (1989)or because of point mutations in the RT gene (Gerard, G. F., et al,FOCUS 14:91 (1992)), can also be used at 50° C., but not at 55° C., tocatalyze cDNA synthesis.

[0236] Therefore, the thermal stability of RNase H⁻ RSV RT and itsutility in higher temperature (i.e., above 50° C.) reverse transcriptionreactions for synthesis of large cDNAs was examined.

[0237] Methods

[0238] Enzymes and RNAs. SuperScript RT (SS RT), SuperScript II RT (SSII RT), and Moloney murine leukemia virus (M-MLV) RT were from LTI. AMVRT was from Seikagaku America, Inc., or was prepared as described abovein Example 4. SS RT is an RNase H⁻ form of M-MLV RT in which RNase Hactivity has been eliminated by removing the RNase H domain of the RTpolypeptide, resulting in an enzyme with a molecular weight of 57 KDarather than 78 KDa (Gerard, G. F., et al., FOCUS 11:66 (1989); Kotewicz,M. L., et al. Nuc. Acids Res. 16:265 (1988)). SS II RT is an RNase H⁻form of M-MLV RT in which RNase H activity has been eliminated by theintroduction of three point mutations in the RNase H domain of M-MLV RT(Gerard, G. F., et al., FOCUS 14:91 (1992)). Rous Associated Virus (RAV)RT was from Amersham. RSV RNase H⁻ and RSV RNase H⁺ RT were cloned,expressed and purified as described above in Examples 1 and 2. The RNAsused as templates were synthetic RNAs of 1.4, 2.4, 4.4 and 7.5 Kb, eachwith a 120 nucleotide poly(A) tail at the 3′ end, obtained from LTI.Synthetic CAT mRNA was from LTI.

[0239] Model System for Determining Functional Thermal Stability ofReverse Transcriptases. A mixture of 1.4-, 2.4-, 4.4-. and 7.5-Kb mRNAswas used to test the ability of various RTs to synthesize full-lengthcDNA copies at various temperatures. The cDNA products synthesized werelabeled radioactively by the RT-catalyzed incorporation of a ³²P-labeleddeoxyribonucleotide triphosphate precursor. The ³²P-labeled cDNAproducts were fractionated by alkaline agarose gel electrophoresis(Carmichael, G. G., and McMaster, G. K., Meth. Enzymol. 65:380 (1980)).The gel was dried and the size distribution of the cDNA products wasestablished by autoradiography. Using the autoradiographic film as atemplate, the full length cDNA bands at 1.4, 2.4, 4.4 and 7.5 Kb werecut from the dried gel and counted in scintillant to establish theamount of each full length product synthesized.

[0240] cDNA synthesis reaction conditions. All cDNA synthesis reactionswere carried out at the indicated temperatures for 30 or 50 minutes. Allreaction mixtures were 20 μl and contained the following componentsunless specified otherwise: 50 mM Tris-HCl (pH 8.4 at 24° C.), 75 mMKCl, 10 mM dithiothreitol, 1 mM each of [³²P]dCTP (300 cpm/pmole), dGTP,dTTP, and dATP, 25 μg/ml p(dT)₂₅₋₃₀, 12.5 μg/ml each of 1.4-, 2.4-,4.4-, and 7.5-Kb mRNA, and 35 units of cloned rat RNase inhibitor. Inaddition, reaction mixtures contained the following: SS II RT: 0.5 mMdNTPs (instead of 1 mM), 3 mM MgCl₂ and 200 units of SS II RT RSV H⁻ RT:7.5 mM MgCl₂ and 21 units of RSV H⁻ RT; AMV RT: 50 mM KCl, 10 mM MgCl₂,4 mM sodium pyrophosphate and 29 units of AMV RT RSV H⁺ RT: 50 mM KCl,10 mM MgCl₂, 4 mM sodium pyrophosphate and 24 units of RSV H⁺ RT RAV RT:50 mM KCl, 10 mM MgCl₂, 4 mM sodium pyrophosphate and 24 units of RAV RT

[0241] Reaction mixtures containing all components except enzyme werepreincubated at the desired temperature for three minutes, and then RTwas added to initiate cDNA synthesis.

[0242] Half Life Determinations. The half lives of RTs were determinedby incubating individual tubes of RT at a desired temperature forappropriate lengths of time and stopping the incubation by placing thetube on ice. RSV RTs, RAV RT and AMV RT were incubated in 20 μl aliquotscontaining 50 mM Tris-HCl (pH 8.4), 75 mM KCl, 7.5 mM MgCl₂, 10 mMdithiothreitol, 50 μg/ml CAT mRNA, 25 μg/ml p(dT)₁₂₋₁₈, and 350-700units/ml RT. Murine RTs (SS RT, SS II RT, M-MLV RT) were incubated inmixtures containing the same components except MgCl₂ was at 3 mM andenzyme was at 2,500 units/ml. An aliquot from each tube (5 μl for avianRTs and 1 μl for murine RTs) was assayed for RT activity in a unit assayreaction mixture to determine residual RT activity.

[0243] UnitAssays. Unit assay reaction mixtures (50 μl) contained 50 mMTris-HCl (pH 8.4), 40 mM KCl, 6 mM MgCl₂, 10 mM dithiothreitol, 500 μM[³H]dTTP (30 cpm/pmole), 0.5 mM poly(A), and 0.5 mM (dT)₁₂₋₁₈. Reactionmixtures were incubated at 37° C. for 10 minutes and labeled productswere acid precipitated on GF/C glass filters that were counted in ascintillation counter.

[0244] Results and Discussion

[0245] With a few exceptions, the half lives in the presence of atemplate primer at 45° C., 50° C., 55° C. and 60° C. were determined forRSV H⁺ RT, RAV RT, AMV RT, RSV H⁻ RT, M-MLV H⁺ RT, SS RT, and SS II RT.The results are shown in FIG. 39 and Table 3. TABLE 3 HALF LIVES OFREVERSE TRANSCRIPTASES¹ HALF LIFE (MINUTES) AT: ENZYME 45° C. 50° C. 55°C. 60° C. RSV H⁻ RT 440 138 5 0.75 RSV H⁺ RT ND² 30 ND² ND³ RAV RT 15937 3.8 ND³ AMV RT 96 16 1.3 ND³ SuperScript II RT 105 7 ND³ ND³SuperScript RT 120 3 ND³ ND³ M-MLV RT 65 2.5 ND³ ND³

[0246] The results shown in FIG. 39 and Table 3 clearly demonstrate thatRNase H⁺ RTs (RS V, RAV and AMV) are much more thermo stable than M-MLVRT, and have reasonable half lives at 50° C. Furthermore, mutating theseRTs to produce their corresponding RNase H⁻ forms further increasestheir half lives. Most dramatically, RNase H⁻ RSV RT had a much longerhalf life than any other retroviral RT at 45° C., 50° C. and 55° C.Thus, introduction of a single amino acid change into the RNase H domainof each subunit of RSV RT increases its half life at 50° C. by nearlyfive-fold.

[0247] The impact of this increased thermal stability on cDNA synthesisat temperatures above 50° C. was found to be dramatic. FIG. 40 shows theresults of a comparison of the performance of these RTs at 45° C., 50°C., 55° C. and 60° C. in copying mRNA of 1.4 to 7.5 Kb in length. Withthe exception of RSV H⁻ RT, none of the RTs were found to producesubstantial product longer than 2.4 Kb in length at 55° C. or 60° C. RSVH⁻ RT, in contrast, continued to make full-length 7.5-Kb cDNA at 55° C.,and 4.4-Kb cDNA at 60° C. FIGS. 41 and 42 show a more detailedcomparison of the two RTs that performed best in FIG. 40 (i.e., SS II RTand RSV H⁻ RT). At temperatures above 55° C., RSV H⁻ RT was found tocontinue to synthesize cDNAs of all lengths, while SS II RT producedonly low levels of cDNA greater than 1 Kb in length.

[0248] Taken together, these results demonstrate that RNase H⁻ RSV RT ismuch more thermoactive than any other retroviral RT availablecommercially, and can be used to synthesize longer cDNAs (up to 4 Kblong) at 60° C. This enhanced thermoactivity of RNase H⁻ RSV RT is dueto an increased thermal stability, relative to RNase H⁺ RT, at 50° C. to60° C., which makes RSV H⁻ RT an ideal enzyme for use in the reversetranscription of mRNA at 50° C. to 60° C.

Example 6 Reverse Transcription with Avian RTs at Elevated Temperatures

[0249] In Example 5 (Table 3 and FIG. 39), the half lives of various RTswere presented. In particular, half lives were reported for cloned RSVRT in which the RNase H domain of each subunit was mutated to eliminateRNase H activity (Example 1; Asp450—Ala in both the a subunit and the βsubunit).

[0250] To further examine the effects of these mutations on RT halflife, constructs were produced as described above, in which only one ofthe two subunits were mutated at one time, such that variouscombinations of mutants were formed (e.g., α RNase H⁻/β RNase H⁺ and αRNase H⁺/β RNase H⁻). The half lives of these RSV RTs, as well as clonedAMV α RNase H⁻/β RNase H⁺ RT, were determined as described above in theMethods section of Example 5.

[0251] As shown in Table 4, when the α subunit in RSV or AMV RT wasmutated, leaving the β subunit wild type intact, the resulting RTdemonstrated greater thermal stability than that observed for otheravian RTs. These mutant RTs were also examined for their functionalthermal stability using the model system described in the Methodssection of Example 5. For each enzyme, the increased thermal stabilitywas found to correlate with improved functional performance—for example,the α RNase H⁻/β RNase H⁺ avian RTs, which demonstrated the highestthermal stability at 55° C. (Table 4), also demonstrated the highestfunctional activity at various elevated temperatures (Table 5). TABLE 4Half Lives of RSV and AMV Reverse Transcriptases Enzyme Half Life(Minutes) at 55° C. RSV αH⁻βH⁻ RT 5 RSV αH⁻βH⁺ RT 7 RSV αH⁺βH⁻ RT 2 RSVαH⁺βH⁺ RT 1.9 AMV αH⁻βH⁺ RT 6 Native AMV RT 1.3 Native RAV RT 3.8

[0252] TABLE 5 Functional Activities of RSV RTs at ElevatedTemperatures. Amount of Full-Length Temperature, Product Produced(pMoles) Enzyme ° C. 1.4 Kb 2.4 Kb 4.4 Kb 7.4 Kb RSV αH⁻/βH⁻ RT 45.0132.9 80.5 56.7 28.1 55.0 115.4 70.7 40.8 12.5 57.5 81.9 43.2 17.2 3.160.0 7.0 1.8 0 0 62.5 0 0 0 0 RSV αH⁻/βH⁺ RT 45.0 145.2 85.3 57.9 31.855.0 161.3 83.0 53.3 21.5 57.5 140.1 77.7 41.1 11.6 60.0 67.6 30.0 7.50.1 62.5 4.1 0.8 0 0

Example 7 Alternative Methods of Generating Avian Reverse Transcriptasesand Characterization of their Properties

[0253] As noted above in the Related Art section, three prototypicalforms of retroviral RT have been studied thoroughly—M-MLV RT, HIV RT,and ASLV RT (which includes RSV and AMV RT). While each of theseretroviral RTs exist as heterodimers of an α and a β subunit, there havebeen no reports heretofore of the simultaneous expression of cloned ASLVRT α and β genes resulting in the formation of heterodimeric αβ RT.

[0254] Examples 1-4 above described the cloning, expression andpurification of αβ forms of RSV and AMV RT that copy mRNA efficiently.Formation of αβ RT was achieved in baculovirus-infected insect cells byco-expression of genes for α and β from a dual promoter vector. Thestudies presented in this Example were designed to generate RSV αβ RT bya variety of other methods that have been used to successfully clone andexpress HIV p66/p51 RT. In addition, as described below, the individualsubunits of RSV RT, including βp4, β and α, have now been cloned,expressed, and purified, and the abilities of these subunits to copymRNA has now been characterized.

[0255] Materials and Methods

[0256] General Methods

[0257] Mutations and plasmid constructions were conducted using standardmolecular biology methods (see e.g., Sambrook, J. et al., MolecularConing: A Laboratory Manual, 2nd Ed., Cold Spring Harbor, N.Y.: ColdSpring Harbor Laboratory Press (1989)), modified as described below.Plasmid preparation, PCR, gel electrophoresis, DNA fragment isolationand cloning, insect cell culture and baculovirus production were allperformed as described for RSV RT cloning and expression in Example 1.

[0258] Cloning and Expression of RSV RT in E. coli

[0259] A number of approaches were tried to generate RSV αβ RT from RSVRT βp4 in E coli.

[0260] PCR of NdeI-XhaIfragment. The amino end of the RSV RT βp4 genewas mutagenized to introduce an NdeI site by PCR (1 cycle 94° C. for 5min.; 15 cycles 94° C., 10 sec, 55° C., 15 sec, 72° C., 15 sec.; 1 cycle72° C., 5 min) of pJD100 (FIG. 7) with the following oligonucleotides:Oligonucleotide #11: (SEQ ID NO:17) 5′-ATT ATT CAT ATG ACT GTT GCG CTACAT CTG GC-3′ Oligonucleotide #12: (SEQ ID NO:18) 5′-TAC GAT CTC TCT CCAGGC CAT TTT C-3′

[0261] The NdeI site in oligonucleotide #11 above appears in bold anditalicized print, while the bases that are underlined inoligonucleotides #11 and #12 were derived from authentic RT genesequences. The PCR product with these two oligos contained an NdeI siteat the beginning of the gene, and retained the Xhal site which ispresent within the RT gene. The PCR product was cloned into pUC18between the NdeI and XbaI sites, forming pUC18 #3.

[0262] Site-directed mutagenesis to introduce a SmaI site to clone p15.A 3 Kb PstI fragment from pJD100 containing the entire RSV RT gene wascloned into M13mp19. Clone RF-SmaI was then produced by introducing aSmaI site at the carboxy end of the RSV RT βp4 gene by site-directedmutagenesis (see Example 1), using the following mutagenicoligonucleotide: Oligonucleotide #13: 5′-ACT CGA GCA GCC CGG GAA CCTTTG-3′ (SEQ ID NO:19)

[0263] Reconstruction of the RTgene. The 2.8 kb SmaI-PstI fragment fromRF-SmaI was cloned into pUC18 at the SmaI/PstI sites. The clone wasdesignated as pUC18-PstI-SmaI. TheNdeI-XbaI fragment from pUC18 #3 wasintroduced into pUC18-PstI-SmaI to regenerate the entire RSV RT βp4 genewith an NdeI site at the initiation codon. The clone was designated aspUC18-RT.

[0264] Cloning entire RT gene in pRE2. The NdeI-XbaI fragment from pUC18#3 was cloned into expression vector pRE2 to generate pRE2-Nde-Xba. pRE2contains an inducible lambda pL promoter. The rest of the RSV RT βp4gene was subcloned as an NdeI-SstI fragment from pUC18-PstI-SmaI intopRE2-Nde-Xba digested with XbaI and SstI, generating pRE2-RT.

[0265] Cloning of p15 into pRE2-RT RSV RT is processed from RSV RT βp4to the αβ heterodimer by the RSV p15 protease. In order to makeauthentic αβ, the p15 protease gene was cloned and expressed with theRSV RT βp4 gene as follows. The RSV p15 protease gene was generated byPCR using pJD100 as target and the protocol described above. Theoligonucleotides used for PCR were as follows: Oligonucleotide #14:5′-AT TAC CCG GGA GG A TAT CAT ATG TTA GCG ATG ACA ATG GAA CAT AAA G-3′(SEQ ID NO:20) Oligonucleotide #15: 5′-A TAT GTC GAC TCA CAG TGG CCC TCCCTA TAA ATT TG-3′ (SEQ ID NO:21)

[0266] In oligonucleotides #14 and #15 above, the restriction sites(SmaI, NdeI and SalI) are indicated in bold letters while the region ofbases underlined is the ribosome binding site. PCR using theseoligonucleotides generated a ˜450 bp fragment, which was digested withSmaI and SalI and cloned into pUC19. The clone was designated aspUC19-p15. The p15 gene was introduced into pRE2-RT by subcloning the450 bp SmaI-SalI fragment at the SmaI/SalI sites. The final plasmid wasdesignated pRE2-RT.p15.

[0267] Expression of RTfrom pRE2-RT and pRE2-RTp15. E. coli CJ374containing either pRE2-RT or pRE2-RT.p15 was grown at 30° C. in EG brothin the presence of ampicillin (100 μg/ml) and chloramphenicol (30 μg/ml)to an A590 of 0.5. Half of the culture was induced at 42° C. for 45min., and then outgrown at 30° C. for 2 hr. The other half was grown at30° C. as an uninduced control. None of the cultures produced anyvisible induced protein upon examination by SDS-PAGE. None of the cellextracts displayed any RT activity.

[0268] Recloning of RTp15 inpRE1. The lack of RT expression in the aboveconstructs suggested that it was possible that (i) a mutation had beenintroduced into the RT gene during PCR to render it inactive, or (ii)during cloning a mutation in the lambda pL promoter arose, since RT isthought to be toxic to E. coli. Thus, the entire RT.p15 gene wasrecloned into pRE1, which was the same as pRE2 except the multiplecloning site was in an opposite orientation, as a NdeI-SalI fragment andthe construct was introduced into E. coli CJ374. In addition, a 2269 bpHpaI-KpnI fragment of the resulting clone was replaced by the sameHpaI-KpnI fragment from pJD 100. This replacement left only about a 200bp amino terminal region derived from PCR. Moreover, this region (NdeIsite to HpaI site) was sequenced to confirm that there was no mutationdue to PCR. The plasmids were designated as pRE1-RT.15. This plasmid wasalso introduced into BL21, a protease-deficient E. coli strain. The p15gene was also deleted from PRE1-RT. 15 by digesting the plasmid withXhoI and SalI and recircularizing the plasmid. The resulting plasmid wasdesignated as pREl-RT.

[0269] Expression of RT in E. coli CJ374 and BL21 harboring pRE1-RT andpRE1-RT15. The cultures were grown as described above. The soluble cellextract was assayed for RT activity. Although the activity was extremelylow, it was clear that the RT activity in the induced cell extract was10-times higher than that in the uninduced cell extract. The level ofactivity was similar in both CJ374 and BL21. The levels of expression ofRT from pREl-RT and pRE1-RT. 15 were similar.

[0270] Cloning of RT gene under a tac promoter. Since RSV RT βp4 was notexpressed well under a lambda pL promoter, expression was attemptedunder the control of a different promoter. The RSV RT βp4 gene with andwithout the p15 gene was cloned under a tac promoter. For cloning the RTgene, pRE1-RT was digested with NsiI, blunt-ended with T4 DNA polymeraseand finally, digested with XhoI. The RT fragment was purified from anagarose gel. For cloning the RT.15 gene, pRE1-RT.15 was digested withNsiI, blunt-ended with T4 DNA polymerase and finally, digested withSalI. The RT.15 gene combination fragment was purified from an agarosegel. The vector pTrc99A (Pharmacia) was digested with NcoI, blunt-endedwith Klenow fragment and finally, digested with SalI. The large vectorfragment was purified and ligated with either purified RT or RT. 15fragment. The resulting constructs were introduced into E. coli DH10B.Clones with correct inserts were saved. The clones were designated aspTrcRT and pTrcRT.15.

[0271] Expression of RT under a tac promoter. E. coli cells harboringpTrcRT or pTrcRT.15 were grown at 37° C. in a buffered-rich medium to anA590 to 0.1 or 0.6 before addition of IPTG (1 mM) for induction of RSVRT βp4 expression. The cells were collected 2 hr after induction. Theuninduced cultures were grown similarly without addition of any inducer.The RT enzyme activity in soluble cell extract was equivalent to thatobtained from constructs with the lambda pL promoter.

[0272] SDS polyacrylamide gel electrophoresis of the expressed proteins.RSV RT is composed of two subunits of molecular mass 94 kD (β) and 62 kD(α). The α subunit is a proteolytic fragment derived from the β subunit.The proteolysis is accomplished by RSV p15 protease in vivo. However,when induced E. coli extracts bearing the plasmids described wereexamined, two induced proteins of molecular mass 75 kD and 62 kD weredetected. Both proteins were found to be mainly in the insolublefraction of E. coli extracts. The presence of a 75 kD and not theexpected 94 kD protein following expression in E. coli suggested that(i) there was a mutation near the carboxy end causing prematuretranslation termination, (ii) there was proteolysis in the E. colicytosol, or (iii) there was internal translation initiation of RT. Whenthe carboxy terminal region of the RT gene was sequenced, no mutationwas found that might have caused premature translation termination. Totest whether there was an internal translation start, the RT gene wascloned as a HpaI-XhoI fragment in pTrc99 digested with SmaI and SalI. Noinduced protein could be detected suggesting that there was no internalstart to generate either a 75 kD or 62 kD protein.

[0273] Expression of RT in a variety of E. coli hosts. As describedabove, expression of RSV RT was examined in E. coli DH10B, CJ374 andBL21. None of these E. coli hosts produced αβ RT, and the level of RTactivity in each host was extremely low. Since M-MLV RT expresses wellin E. coli N₄₈₃O and HIV RT expresses well in E. coli RR1, the levels ofexpression of RSV RT in these two hosts were examined. Neither of thesehosts was found to express active RT any better than the other hoststested, nor did they produce any full length 94 kD protein.

[0274] Expression of RT as a fusion protein. It is now well establishedthat some proteins that do not express well in E. coli are betterexpressed as a translation fusion, in which the protein from a wellexpressed gene forms the amino end of the fusion protein. The gene ofone such fusion partner, thioredoxin, is present in a vector calledpTrxfus (Genetics Institute; Cambridge, Mass.). The level of expressionof RSV RT βp4 fused with thioredoxin was therefore examined.

[0275] In the course of expressing RSV RT in a baculovirus system, theRSV RT gene fragment was cloned as a SmaI-XhoI fragment in pBacPAK9 toconstruct pBacPak-RT (see below). To make the thioredoxin fusion,pBacPak-RT was digested with XmaI (same recognition as SmaI) and PstIand the RT fragment was purified on an agarose gel. The vector pTrxfuswas digested with XmaI and PstI and the large vector fragment waspurified. The purified fragments were ligated and E. coli CJ374 andGI724 or GI698 (Genetic Institute, MA) were transformed with the ligatedmaterial. Clones with the correct insert were saved. When cultures wereinduced and the cellular extracts were assayed for enzyme activity, noRT activity was detected. Both induced cultures, however, produced alarge amount of insoluble fusion protein of the expected size as judgedby SDS-PAGE of the extracts. Other fusions were also tested. The RSV RTβ gene was fused to the GST (glutathione S transferase) gene, and theRSV RT α gene was fused to the lambda CRO protein gene. Expression ofeither fusion from the trc promoter in E. coli strain DH10B resulted ina large amount of insoluble protein of the appropriate molecular weight,but little RT activity was detected in extracts of induced cells. Anexpression vector with both fusions (GST-beta and CRO-alpha) wasconstructed in which the fusions were co-expressed by induction of thetrc promoter. Co-expression of both fusions in E. coli strain DH10Bresulted in large amounts of insoluble protein of the appropriatemolecular weights, but little RT activity.

[0276] Cloning and Expression Separately of Genes Encoding RS VRTSubunits α, β, and βp4 in a Baculovirus System

[0277] Cloning of RSV RT βp4 in a Baculovirus System

[0278] To clone the RSV RT βp4 gene in the baculovirus transfer vector,pBacPAK9 (Clontech), a fragment of the RSV RT gene was generated by PCR.To facilitate this PCR, the following oligonucleotide was designed witha BamHI site (bold) and the ATG initiation codon at the beginning of theRSV βp4 gene: Oligonucleotide #16: 5′-TAT TAG GAT CCC ATG  ACT GTT GCGCTA CAT CTG GC-3′ (SEQ ID NO:22)

[0279] Oligonucleotides #12 and #16 (SEQ ID NOs: 18 and 22,respectively) were used for PCR using pJD100 as template. The PCRproduct was digested with BamHI and XbaI, and then ligated to pBacPAK9digested with BamHI and XbaI. One of the clones, pBP-RT(PCR) (FIG. 43),was used for further cloning. To reconstitute the entire RT gene, thesmall HpaI-XhoI fragment of pBP-RT(PCR) was replaced with the 2500 bpHpaI-XhoI fragment of pRE1-RT.15. The reconstituted plasmid wasdesignated as pBP-RT(ATG) (FIG. 44).

[0280] Cloning of p15 in pBacPAK-RT(ATG). Placing the RSV p15 proteasegene in pBP-RT(ATG) was achieved by a three-step cloning. First, a p15gene fragment was subcloned from pUC19-p15 (see above) into pSport1(LTI) as a KpnI-SalI fragment to generate pSport-p15. Second, the p15fragment was subcloned from pSport-p15 as a NotI-SmaI fragment into abaculovirus transfer vector, pAcUW43, to generate pAcUW43-p15. Thiscloning was done to introduce the p15 gene under a p10 promoter.Finally, the p15 gene including the p10 promoter was subcloned frompAcUW43-p15 as a XhoI-NotI fragment into pBP-RT (ATG) to generatepBP-RT15(ATG) (FIG. 45).

[0281] Expressing RSV RT βp4 in insect cells. Insect cells (SF9) weretransfected with a mixture of pBP-RT15(ATG) and linearized baculovirusvector DNA (BaculoGold, Pharmingen). In this system, recombinationbetween the pBP-RT15(ATG) plasmid and the baculoviral DNA results inrecombinant viral genomes with the RSV RT βp4 gene downstream of thepolyhedrin promoter. Recombinant virus was isolated from the supernatantby standard techniques and viral stocks were made, one of which waschosen for further study. Insect cells were infected with pure viralstock and infected cells were harvested at various times afterinfection. RT activity was easily detectable in the infected cells.

[0282] Generation of an RNase H− RSV RT The generation of a mutation inthe RNase H region of the RSV RT βp4 gene is described in detail abovein Example 1. To introduce the mutation into pBP-RT(ATG), the HpaI-KpnIfragment of pBP-RT(ATG) was replaced with the HpaI-KpnI fragment ofM13RTH− (FIG. 13). The newly constructed plasmid was designated aspBP-RT(H−) ATG. Insect Cells (Sf9) were transfected with a mixture ofpBP-RT(H−) and linearized baculovirus vector DNA (BaculoGold,Pharmingen). Recombination between the pBP-RT(H−) plasmid and thebaculoviral DNA results in recombinant viral genomes with the RSV RTβH−P4 gene downstream of the polyhedrin promotor. Recombinant virus wasisolated from the supernatant by standard techniques. Insect cells wereinfected with viral stock and infected cells were harvested at varioustimes after infection. RT activity was easily detectable in the infectedcells.

[0283] Attaching a histidine tag to RSV RT βp4. pBP-RT(H−) was cleavedwith BamHI and XbaI and the 0.9 kb fragment with the amino end of theRSV RT βp4 gene was cloned into pFastBacHT at the BamHI-XbaI sites,creating (by translational fusion) a βp4 partial construct with ahistidine tag at the amino end (pFBHTβP4). The 2 kb XhaI-YhoI fragmentwith the carboxy end of RSV RT ⊕H−p4 from pBP-RT(H−) was inserted intothe XbaI-XhoI sites of pFBHTβP4, creating pFBH−TβP4. pFBH−TβP4wastransformed into E. coli DH10Bac cells and the His-tagged RSV RT βH−p4gene was transposed to bacmid. Bacmid DNA was isolated and transfectedinto SF9 insect cells. Viral preparations from infected cell cultureswere used to infect SF21 cells, and His- tagged RSV RT βH−p4 frominfected cells was isolated and characterized.

[0284] Transfection of insect cells. The transfection of Sf9 cells wasdone using Baculogold virus (Pharmingen, Callif.) and pBP-RT(H−)ATG togenerate recombinant virus. Ten wells on two 6-well (60 mm) tissueculture plates were seeded 1×10⁶ Sf9 cells (LTI). The cells were allowedto attach for 30 min at 27° C. While the cells were attaching, ten tubeseach containing 200 μl of Sf-900II SFM medium (LTI) were set up. In tube1, 500 ng of Baculogold and 2 μg of pBP-RT(H−)ATG were added. In tube 2,250 ng of Baculogold and 1 μg of pBP-RT(H−)ATG; and tube 3, 125 ng ofBaculogold and 500 ng of pBP-RT(H−)ATG were added. Tubes 6 through 9contained 36 μl oflipofectin (LTI). Contents of tube 1, tube 2, tube 3,tube 4 and tube 5 were transferred to tubes 6, 7, 8, 9 and 10,respectively. Into each of these tubes, 2 ml of Sf-900 II SFM wereadded. The culture medium from each well was removed and 2 ml of theDNA/lipofectin mix was dispensed in two wells, 1 ml each. Thus, thewells containing the mixture of tubes 1 and 6, 2 and 7, and 3 and 8contained DNA mixtures at different amounts; the wells containing themixtures of tubes 4 and 9 were controls containing lipofectin but noDNA; and the wells containing the mixtures of tubes 5 and 10 werecontrols containing neither DNA nor lipofectin. The plates wereincubated at 27° C. for 4 hr, the medium was removed and replaced with 4ml fresh Sf-900 II SFM, and the plates were incubated for an additional72 hrs at 27° C. The phage supernatants from the DNA containing wellswere removed and marked as primary phage stocks. A second infection wasdone by infecting 1×10⁶ Sf9 cells with 1 ml of the primary phage stockto amplify the recombinant phage. The plates were incubated for 48 hrsat 27° C. and the phage stocks were collected.

[0285] Injection of insect larvae. Trichoplusa ni larvae were injectedwith recombinant virus bearing pBP-RT(H−)ATG and the larvae wereharvested as described (Medin, J. A., et al., Methods in MolecularBiology 39:26 (1995)).

[0286] Expression of RSV RT αH− in a Baculovirus System

[0287] pDA (FIG. 19) was transformed into E. coli DH10Bac cells, the RTαH− gene was transposed to bacmid, and the bacmid DNA was purified andtransfected into SF21 insect cells. Viral preparations from infectedcell cultures were used to infect SF21 cells, and RSV RT αH− frominfected cells was isolated and characterized.

[0288] Expression of RSV RT βH−His in a Baculovirus System

[0289] pDABH−His (FIG. 21) was cleaved with RsrII and PstI, and the 2.6kb fragment with the βH−His gene was cloned into the RsrII-PstI sites ofpFastBac. PFBBH−His was transformed into E. coli DH10Bac cells, the RTβH−His gene was transposed to bacmid, and the bacmid DNA was purifiedand transfected into SF21 insect cells. Viral preparations from infectedcell cultures were used to infect SF21 cells, and RSV RT βH−His frominfected cells was isolated and characterized.

[0290] Cloning and Expression of Genes Encoding RSV αβ RT in which thePolymerase Active Site is Mutated

[0291] Generation ofRSVRTs mutated in the polymerase domain. Alignmentof the RSV RT peptide sequence with sequences from HIV RT, M-MLV RT, andsequences of other RT genes revealed the probable location of one of thecatalytic residues in the RSV RT polymerase domain, D110 (aspartic acidreside at position number 110). According to the literature, mutation ofthe corresponding amino acid in the larger chain of. HIV RT from D(aspartate) to E (glutamic acid) resulted in nearly total loss ofpolymerase activity. Single strand DNA was isolated from pJB-His byinfection of E. coli DH5αF′IQ/pJB-His cells with M13KO7, and this DNAwas mutated by site-directed mutagenesis (see Example 1 for detailedprotocol) with the following oligonucleotide: Oligonucleotide #17: (SEQID NO:23) 5′- GCAATCCTTGAGCTCTAAGACCATCAGGG 3′

[0292] This oligonucleotide induces the mutation of the aspartateresidue at position #110 or the RSV RT catalytic domain to glutamate(D110E) and adds an SstI site (bold), forming plasmid pJBD110E-His (FIG.48). This mutated site was introduced into the RSV RT a gene byinserting the 460 bp NheI-Eco471II fragment from pJBD110E-His into the6.5 kb fragment ofNheI-Eco471II cleaved pDA, forming pDAD110E (FIG. 49).The D110E mutation was also introduced into the β gene by inserting the460 bp NheI-Eco471II fragment into NheI-Eco471II cleaved pFBBH−His (FIG.46), forming pFBBD110E-His (FIG. 50). The 2.6 kb RSV RT βD110E gene wascloned from pFBBD110E-His into pDABHis (FIG. 51) as a 2.6 kb RsrII-EcoRIfragment, replacing the RSV RT β-His gene, forming pDABD110EHis (FIG.52). pDABHis was cleaved with XhoI+PvuI, and the 4.6 kb fragment withthe β gene was joined to the 4.9 kb pDAD110EXhoI-PvuI fragment, formingpDAD110EBHis (FIG. 53). The 4.6 kb pDABD110EHis XhoI—PvuI fragment withthe βD110E gene was joined to the 4.9 kb pDAD110E XhoI-PvuI fragment,forming pDAD110EBD110E (FIG. 54) (which has a his tag despite itstruncated name). PDAD110EBHis, pDABD110EHis, and pDAD110EBD110E weretransformed into E. coli DH10B-Bac, the RT genes were transposed to thebacmid, and the bacmid DNA was purified and transfected into SF21 insectcells. Viral preparations from infected cell cultures were used toinfect SF21 cells, and RSV RT from infected cells was isolated andcharacterized.

[0293] The three mutant plasmids are summarized in the following table,where “w.t.” refers to the wild type amino acid at the 110 position (D,aspartic acid) and D110E refers to the mutation at the 110 position (Dto E, glutamic acid): Plasmid α β pDABHis w.t w.t. pDABD110EHis w.tD110E pDAD110EBHis D110E w.t. pDAD110EB110E D110E D110E

[0294] Cloning and Expression of RSV ⊖p4 RT in Yeast

[0295] Cloning of the RSV βp4 RT gene. The pHIL-D2 vector available fromInVitrogen (CA) was digested with EcoRI, blunt ended with Klenowfragment and treated with alkaline phosphatase. PRE1-RT.15 was digestedwith NdeI and blunt ended with Klenow fragment. The NdeI digestgenerated the entire RSV RT gene without the p15 protease gene. Thefragments were ligated and E. coli DH10B was transformed with ligationmixture. The correct clones were selected for proper insert andorientation. Two of the 8 clones tested had the RT gene fragment in thecorrect orientation. One of the clones, pHILD2-RT, was used for furtherexperimentation. To introduce the RNase H− domain in this plasmid,pHILD2-RT was digested with BamHI plus XhoI and the wild type fragmentwas replaced with BamHI-XhoI fragment from pBacPAK-RT(H—)ATG. The finalclone was screened with SstII. The clone was designated aspHILD2-RT(H−).

[0296] Transformation of Pichia pastoris. Pichia pastoris GS 115(InVitrogen) was used for transformation according to the protocolrecommended by InVitrogen. The plasmids, pHILD2-RT and pHILD2RT(H−),were digested with NotI, phenol-chloroform extracted and ethanolprecipitated before transformation. The transformation yielded 20 clonesfor wild type RSV RT and 12 clones for RSV H− RT in the regenerationplates. These clones were screened for their growth in methanolcontaining plates. Two putative clones were selected from initial 20clones (wild type RT). One of these two, H1, was completely incapable ofgrowing in methanol, and the other, H2, was capable of growing veryslowly in methanol. For the RSV H− RT clones, three clones were selectedout of 12 screened. One of the clones, H-3, grew very slowly inmethanol. The others, H-4 and H-5, grew moderately in methanol. ClonesH1 and H-3 were chosen for expression studies.

[0297] RSV βp4 RT expression in Pichia pastoris. Clones H1 and H-3 weregrown and induced essentially as described in the user's manual providedby the manufacturer (InVitrogen). As a control, GS115 containing aβ-galactosidase gene (InVitrogen) was grown and induced side by side.While no RT activity could be detected in the GS 115/β-gal cells, anappreciable level of activity was detected in both H1 (wild type RT) andH-3 (H− RT) cells. In addition, the activity increased with increasedtime of induction.

[0298] Isolation of RSV ββ, α and βp4βp4

[0299] RSV ββ RT isolation. RSV ββ RT was purified as described inExample 2 for RSV αβ RT with the following exceptions. RSV ββ RT elutedfrom the AF-Heparin-650M column from 55-62% Buffer T, and from the MonoS HR 5/5 column from 58-62% BufferJ. RSVPPRT was 90% homogeneous asjudged by SDS-PAGE.

[0300] RSV α RT isolation. RSV α RT was purified as described in Example2 for RSV αβ RT with the following exceptions. The Chelating SepharoseFast Flow column was equilibrated with 100% Buffer A and washed with100% Buffer A after the clarified crude extract was passed over thecolumn. RSV α RT was eluted with a 10-column volume linear gradient of100% Buffer A to 75% Buffer A+25% Buffer B. The peak fractions of RTactivity from the Chelating Sepharose Fast Flow column (10-12% Buffer B)were pooled, dithiothreitol and EDTA were added to the pool to achievefinal concentrations of 1 mM and 0.1 mM, respectively, and the pool wasdialyzed overnight against 95% Buffer S+5% Buffer T. The dialyzed poolwas loaded on a 22 ml AF-Heparin-650M column equilibrated in Buffer S.After a wash with 9 column volumes of 95% Buffer S+5% Buffer T, thecolumn was eluted with a 9 column volume linear gradient of 95% BufferS+5% Buffer T to 60% Buffer S+40% Buffer T. The peak fractions of RTactivity (11-20% Buffer J) were pooled and dialyzed for 3 to 4 hoursagainst 97.5% Buffer H and 2.5% Buffer J. The dialyzed pool was loadedunto a 3.5 ml phosphocellulose column (Whatman) equilibrated in 100%Buffer H. After a wash with 12 column volumes of 97.5% Buffer H+2.5Buffer J, the column was eluted with a 14 column volume linear gradientof 97.5% Buffer H+2.5% Buffer J to 60% Buffer H+40% Buffer J. The peakfractions of RT activity (12-20% Buffer J) were pooled and dialyzedovernight against 99% Buffer S+1% Buffer T. The dialyzed pool was loadedunto a Mono S Hk 5/5 column equilibrated in Buffer S. After a wash with10 column volumes of 100% Buffer S, the column was eluted with a 20column volume linear gradient of 100% Buffer S to 75% Buffer S+25%Buffer T. The RT peak fractions (15-17% Buffer T) were pooled, dialyzedagainst Storage Buffer overnight, and stored at −20° C. RSV α RT wasfound to be 80% homogeneous as judged by SDS-PAGE.

[0301] RSV βp4βp4 RT isolation. RSV βp4βp4 RT was purified as describedin Example 2 for RSV αβ with the following exceptions. The pooled RTfractions from the Chelating Sepharose Fast Flow column were dialyzedovernight against 90% Buffer H+10% Buffer J. The RSV βp4βp4 RTprecipitated in this buffer. The RT was recovered by centrifugation,dissolved in Storage Buffer containing 0.5 M KCl, and stored at −20° C.RSV βp4βp4 RT was found to be >95% homogeneous as judged by SDS-PAGE.

[0302] Estimation of the Amounts of α, αβ and ββ RSV RT inBaculovirus-Infected Insect Cells

[0303] Chromatography methods described in Example 2 can be used toseparate and isolate any α, αβ and ββ RSV RT present in crude extractsfrom virus-infected insect cells. RSV RT α is separable from the othertwo enzymes forms by chromatography on a Chelating Sepharose Fast Flowcolumn, where α either does not bind or elutes at <30 mM imidazole, andαβ and ββ elute together at >50 mM imidazole. This is possible by virtueof the His₆ tag present on β, but not on α. RSV αβ and ββ RT areseparable subsequently by chromatography on Heparin-650M (αβ elutes at0.45M KCl and ββ elutes at 0.58M KCl). Reverse transcriptase isquantitated by assay with poly(C)·oligo(dG) (Gerard, G. F., et al,Biochemistry 13: 1632 (1974)). The specific activities of RSV RT α, αβand ββ with poly(C)·oligo(dG) are 140,000, 90,000 and 6,000 units/mg ofprotein, respectively.

[0304] Cloning, Expression, Purification and Use of RSV Viral Proteasep15

[0305] RSV viral protease was cloned and expressed as a linked dimer inE. coli and purified from inclusion bodies as described (Bizub, D., etal., J. Biol. Chem. 266: 4951 (1991)).

[0306] Reaction mixtures (25 μl) used to digest RSV βp4 RT with RSVprotease contained 100 mM NaPO₄ (pH 6.0 to 7.0), 1 mM 2-mercaptoethanol,0.01% (W/V) Triton X-100, 2.4 M NaCl, 5 μg RSV βp4 RT, and 5 μg RSVprotease. Incubations were for 1 to 16 hours at 4° C. Digestion productswere analyzed by SDS-PAGE and by assay for recovery of RT activity.

[0307] Assay of RNase H Activity of RSV RT

[0308] The RNase H activity of RSV RT was determined by monitoring thesolubilization of [³H]poly(A) in [³H]poly(A)·poly(dT). Reaction mixtures(50 μl) contained 50 mM Tris-HCl (pH 8.4), 20 mM KCl, 10 mM MgCl₂, 10 μMeach of [³H]poly(A) (300 cpm/pmole) and poly(dT) in [³H]poly(A)·poly(dT)and 10 mM dithiothreitol. Reaction mixtures were incubated at 37° C. for20 minutes. Incubations were terminated by the addition of 80 μl of 20%(W/V) TCA and 10 μl of 1 mg/ml tRNA, and after centrifugation the amountof [³H]poly(A) solubilized was determined by counting the supernatant inaqueous scintillation fluid. One unit of RNase H activity was the amountof enzyme that solubilized one nmole of [³H]poly(A) in 20 minutes at 37°C.

[0309] Assay of DNA Polymerase Activity with Poly(C)·Oligo(dG)₁₂₋₁₈

[0310] The RNA-dependent DNA polymerase activity of RSV RT wasdetermined by monitoring the synthesis of acid insoluble [³H]poly(dG)form poly(C)·oligo(dG). Reaction mixtures (50 μl) contained 50 mMTris-HCl (pH 8.4), 50 mM KCl, 10 mM MgCl₂, 0.5 mM poly(C), 0.2 mMoligo(dG)₁₂₋₁₈, 0.5 mM [³H]dGTP (40 cpm/pmole), and 10 mMdithiothreitol. Reactions were incubated at 37° C. for 10 minutes andlabeled products were acid precipitated on GF/C glass filters that werecounted in a scintillation counter. One unit of DNA polymerase activitywas the amount of enzyme that incorporated one nmole of [³H]dGTP in 10minutes at 37° C.

[0311] Results and Discussion

[0312] Alternative Methods of Generating RSV αβ RT

[0313] Examples 1 through 6 demonstrated that RNase H⁻ forms of avian RTare more efficient than RNase H⁺ RT in copying mRNA. The studiespresented in this Example were designed to determine the efficiency ofmRNA copying by RSV RNase H⁻ αβ RT that was generated by co-expressionof the RSV α and β genes in several expression systems.

[0314] Expression in E.coli. Small amounts of soluble and active α, ββ,βp4βp4, and αβ RSV RT have been purified from E.coli (Alexander, F., etal., J. Virol. 61: 534 (1987); Weis, J. H., and Salstrom, J. S., U.S.Pat. No. 4,663,290 (1987); Soltis, D. A. and Skalka, A. M., Proc. Nat.Acad. Sci. USA 85:3372 (1988); and Cherhov, A. P., et al. Biomed Sci. 2:49 (1991)). However, most of the RSV RT expressed in E. coli in theseprevious reports was in an insoluble form.

[0315] The present efforts to express amounts of RSV RT proteins easilypurified from E coli are documented in the Materials and Methods sectionof this Example. In general, similar results to those previouslypublished were obtained; that is, most of the RSV RT protein expressedin E. coli was insoluble, and only small amounts of RT activity wereobserved. Because of low RT levels, no attempts were made to purify RSVRT expressed in E. coli.

[0316] Expression of the RSV RT βp4 or β gene in cultural insect cells.Heterodimeric p66/p51 HIV RT has been produced in E. coli and yeast hostcells when the cloned gene for HIV p66 was expressed (Lowe, D. M., etal., Biochemistry 27:8884 (1988); Muller, B., et al., J. Biol. Chem.264:13975 (1989); and Barr, P. J., et al., BioTechnology 5:486 (1987)).Formation of the heterodimer occurs by proteolytic processing of p66/p66by endogenous host proteases. In contrast, expression of the gene forHIV RT p66 in cultured insect cells yielded only p66/p66 homodimer(Kawa, S., et al., Prot. Expression and Purification 4:298 (1993)).

[0317] In the present studies, expression of the gene for RSV RT βp4 incultured insect cells was similarly found to result exclusively in theproduction of homodimer βp4βp4 (data not shown); little processing to αβor α was observed. When the RSV RT β gene was expressed in these cells,however, all three forms of RSV RT were produced (Table 6). Most of theRT present in the cells was ββ (50-80%); a small amount of αβ wasproduced (˜10%); and even α was obtained (10-40%). These results suggestthat endogenous host proteases in cultured insect cells proteolyze ββbut not βp4βp4, to αβ. The RSV αβ RT generated by proteolysis of ββ hadmuch lower functional activity than αβ generated by co-expression of theRSV RT α and β genes (Table 7). TABLE 6 Expression Levels of RSV RTs inInsect Cells Infected With RSV RT β Gene Amount of RT Present per 30grams of Insect Cells RSV ββ RT RSV αβ RT RSV α RT Infection No. mg % mg% mg % 1 0.7 54 0.1 8 0.5 38 2 0.12 79 0.017 11 0.015 10 3 0.55 83 0.058 0.06 9

[0318] Expression of RSV RT βp4 in insect larvae. Baculovirus bearingthe RSV RT βp4 gene was also used to infect live larvae by physicalinjection of virus. The level of proteases present in larvae and inlarval extracts is much higher than in cultured insect cells. Processingof βp4 to multiple forms of proteolyzed RT was observed, includingprocessing to α. The major species of RT that could be purified fromthese extracts had four major bands that migrated on SDS-PAGE at 97 kDa(His-tagged β), 87 kDa (proteolyzed β), 67 kDa (partially processedHis-tagged α), and 62 kDa (α with His tag removed by proteolysis). ThisRSV RT had a specific activity of 55,000 units/mg protein, comparable toRSV αβ RT. In the functional activity assay (Example 3), however, the RTpurified from larvae had 85% and 60% of the total product and fulllength product functional activity, respectively, of RSV αβ RT generatedby co-expression of α and β (Table 7). TABLE 7 Specific and FunctionalActivities of Various Forms of RSV RTs Expressed in Insect Cells RT FormIsolated RSV α RT RSV βp4βp4 RT RSV αβ RT RSV ββ RT FunctionalFunctional Functional Activity^(b) Functional Activity Activity SpecificFull- Specific Activity Specific Full- Spec. Full- Gene(s) Activity^(a)Total length Activity Total Full-length Activity Total length Act. Totallength Expressed (U/mg) (ng/μg)^(c) (ng/μg)^(d) (U/mg) (ng/μg) (ng/μg)(U/mg) (ng/μg) (ng/μg) (U/mg) (ng/μg) (ng/μg) α and β 53,191 4,092 874NP^(e) NP NP ND^(f) ND ND NP NP NP β 25,113 1,098 116 15,819 584 41 NDND ND NP NP NP βp4 NP NP NP NP NP NP NP NP NP 15,984 450 67 α NP NP NPNP NP NP 48,759 272 6 NP NP NP

[0319] Co-expression of RSV RT βp4 and RSVprotease p15 in culturedinsect cells. Heterodimeric p66/p51 HIV RT has been produced efficientlyin E. coli by co-expression of HIV protease and HIV RT p66 (Mizuahi, V.,et al, Arch. Biochem Biophys. 273:347 (1989) and Le Grice, S. F. J. andGruninger-Leitch, F., Eur. J. Biochem. 187:307 (1990)). Co-expression ofthe viral protease increased the overall efficiency of convertingp66/p66 to heterodimer. As described above in the Materials and Methodssection of this Example, co-expression of RSV RT β and RSV protease p15genes in E. coli did not result in enhanced production of RSV αβ RT.Similarly, co-expression of RSV RT βp4 and RSV protease p15 in culturedinsect cells did not appreciably enhance the formation of RSV αβ RT.

[0320] Processing of RSV RT βp4 with RSV protease p15 in vitro.Heterodimeric p66/p51 HIV RT has been produced from p66 purified from E.coli by treatment with HIV protease in vitro (Chattopodhyay, D., et al.,J. Biol. Chem. 267:14227 (1992)). Purified RSV RT βp4 was treated withRSV protease p15 to generate RSV αβ RT. This approach was successful ingenerating some αβ from βp4 based upon SDS-PAGE analysis, but severaldifficulties were encountered. First, contrary to what was observed withviral protease treatment of HIV p66 RT, processing of βp4 to αβ did notalways stop at αβ, as α in excess of β was formed as proteolysisproceeded. Second, significant loss of DNA polymerase activity wasobserved during proteolysis, suggesting RSV RT was partially inactivatedby the acid pH reaction conditions required by RSV protease.

[0321] Processing of RSV RT βp4 with chymotrypsin in vitro.Heterodimeric p66/p51 HIV RT has also been produced from p66 by limitedproteolysis with α-chymotrypsin (Lowe, D. M., et al., Biochemistry27:8884(1988)). This approach was tried unsuccessfully with RSV RT βp4.We found the digestion of βp4 with α-chymotrypsin was difficult tocontrol, and proteolysis was observed to not stop at αβ, but to proceedto conversion of βp4 to α.

[0322] Mixing of RSV RT α and β (in vitro) to generate αβ. Heterodimericp66/p51 HIV RT has been produced by mixing separate crude cell lysatescontaining p51 alone and p66 alone (Stahlhut, M., et al., ProteinExpression and Purification 5:614 (1994)). Mixing of the separatesubunits results in formation of a 1:1 molar complex of p66/p51. Incontrast, mixing of purified RSV α RT with purified RSV β RT atapproximately a 1:1 molar ratio did not result in the formation of an αβcomplex. These results are consistent with the notion that the RSVsubunits, once folded separately in an active conformation, prefer toremain separate when mixed.

[0323] Relative Ability of Various Forms of RSV RT to Copy RNA

[0324] A comparison was made of the ability of four different forms ofRSV RT (αβ, ββ, α, and βp4βp4) to copy RNA. The RNase H active site ofeach subunit in these enzymes was mutated to eliminate RNase H activity.Each RT was expressed in cultured insect cells and purified by methodsdescribed above and in Example 1. Two RNAs were used for comparison:synthetic homopolymer poly(A) and 7.5-Kb mRNA. With poly(A)·oligo(dT) astemplate-primer, a specific activity was calculated by determining aninitial rate of poly(dT) synthesis catalyzed at limiting enzymeconcentration, and then normalizing the rate to a given mass (mg) of RTin the reaction. This specific activity simply represents the ability ofa given RT to incorporate a single deoxynucleotide with an artificialtemplate, and does not necessarily represent the ability of the enzymeto copy heteropolymeric RNA. With 7.5-Kb RNA as template, the ability ofthe RTs to make a full-length copy of a long heteropolymeric RNA wasassessed (see Example 3 for details). The results are shown above inTable 7.

[0325] Two different forms of αβ were characterized in Table 7. One formwas generated as the result of the expression of the RSV RT β gene andsubsequent proteolytic processing in host insect cells, and had reducedspecific and functional activity. The other form of αβ was generated byco-expression of the RSV RT α and β genes. This form of αβ had a similarspecific activity to α, approximately 50,000 units/mg, and had a higherspecific activity than either ββ or βp4 pp4 (approximately 16,000units/mg). Comparison of the functional activity of this αβ to other RTforms showed a much more dramatic contrast. RSV αβ RT produced 7, 9 and15 times more total cDNA per mass of enzyme than ββ, βp4βp4 and α,respectively, from 7.5-Kb RNA. Even greater differences were observedwhen yield of full length product was assessed: RSV αβ RT produced 21,13 and 146 times more full length product per mass of enzyme than ββ,βp4βp4 and α, respectively. RSV αβ RT produced by co-expression of theRSV RT α H⁻ and βH⁻ genes is therefore much more efficient in copyingmRNA than is any other form of RSV RT prepared by analogous methods.

[0326] Evidence that Only the α Subunit in RSV αβ RT is Active

[0327] Selective DNA polymerase active site mutagenesis of the HIV RTheterodimer p66/p51 has shown that only the DNA polymerase active siteof p66 is crucial for DNA polymerase activity (LeGrice, S. F. J., etal., The EMBO Journal 10: 3905 (1991) and Hostomsky, Z., et al., J.Virol. 66: 3179 (1992)). The p51 subunit in the HIV p66/p51 heterodimerapparently assumes a conformation which does not have a substratebinding cleft and therefore does not participate directly in dNTPbinding and incorporation (Jacob-Molina, A., et al., Proc Natl. Acad SciUSA 90: 6320 (1993)).

[0328] In the present studies, the same question was asked for RSV αβRT. In this case, since each subunit contains both a DNA polymerase andRNase H active site, combinations of DNA polymerase mutants alone andRNase H mutants alone were characterized. The results are shown in Table8. TABLE 8 DNA Polymerase and RNase H Activities of Various Forms of RSVRT. Specific Activity (Units/mg) Ratio of DNA RSV RT Form DNA PolymeraseRNase H Polymerase/RNase H αH⁺/βH⁺ 52,095 924 56.4 αH⁻/βH⁻ 53,191 <0.1 —αH⁺/βH⁻ 48,250 760 64.5 αH⁻/βH⁺ 50,909 4.5 11,313 βH⁻/βH⁻ 15,819 <0.1 —αH⁻ 48,759 <0.1 — αpol⁺/βpol⁺ 52,095 924 56.4 αpol⁻/βpol⁻ 50 600 0.08αpol⁺/βpol⁻ 57,500 900 63.9 αpol⁻/βpol⁺ 1,143 629 1.82

[0329] As shown in Table 8, when the RNase H active site in bothsubunits was mutated to eliminate RNase H activity, RNase H activity inpurified enzyme was reduced to below detectable levels, while DNApolymerase activity was unchanged. When the RNase H active site in β wasaltered with the same mutation and the α RNase H active site was wildtype, the enzyme was similar to wild type in polymerase and RNase Hactivity. In contrast, mutation of the α subunit in RNase H, but not theβ subunit, resulted in a 200-fold reduction in RNase H activity.Therefore, in the case of RSV αβ RT, the RNase H domain of the largesubunit (β) folds in an inactive conformation. Examination of DNApolymerase active site mutants in RSV αβ RT revealed the same results(Table 8). The α subunit, but not the β subunit, supplies the DNApolymerase catalytic activity. So, in contrast to HIV p66/p51 RT, thesmaller RSV αβ RT subunit, not the larger, maintains enzymatic activity.The common element in both enzymes is that the subunit possessing DNApolymerase and RNase H domains folds in an active conformation, whilesubunits missing the RNase H domain or possessing an additional domain(integrase), fold in an inactive conformation.

[0330] In sum, these results demonstrate that only the αβ form of ASLVRT copies mRNA efficiently. In addition, the present results indicatethat expression of ASLV RT in E. coli results in little or no productionof active RT, while expression and activity increase dramatically bycloning and expression of ASLV RT in insect cells and yeast. Finally, byplacing point mutations in either the DNA polymerase or the RNase Hactive site of the α or β subunit of RSV αβ RT, it has now beendiscovered that the DNA polymerase and RNase H catalytic activities ofthe RSV RT αβ heterodimer reside in the a subunit alone.

[0331] Having now fully described the present invention in some detailby way of illustration and example for purposes of clarity ofunderstanding, it will be obvious to one of ordinary skill in the artthat the same can be performed by modifying or changing the inventionwithin a wide and equivalent range of conditions, formulations and otherparameters without affecting the scope of the invention or any specificembodiment thereof, and that such modifications or changes are intendedto be encompassed within the scope of the appended claims.

[0332] All publications, patents and patent applications mentioned inthis specification are indicative of the level of skill of those skilledin the art to which this invention pertains, and are herein incorporatedby reference to the same extent as if each individual publication,patent or patent application was specifically and individually indicatedto be incorporated by reference.

1 24 1 39 DNA Artificial Sequence Oligonucleotide 1 auggagaucucucatatgac tgttgcgcta catctggct 39 2 37 DNA Artificial SequenceOligonucleotide 2 aacgcguacu agugttaaca gcgcgcaaat catgcag 37 3 36 DNAArtificial Sequence Oligonucleotide 3 cuacuacuac uaggtaccct ctcgaaaagttaaacc 36 4 45 DNA Artificial Sequence Oligonucleotide 4 caucaucaucauctcgagtt atgcaaaaag agggctcgcc tcatc 45 5 36 DNA Artificial SequenceOligonucleotide 5 ggacccactg tctttaccgc ggcctcctca agcacc 36 6 39 DNAArtificial Sequence Oligonucleotide 6 caucaucauc aucccgggtt aatacgcttggaaggtggc 39 7 35 DNA Artificial Sequence Oligonucleotide 7 cuacuacuacuatcatgact gttgcgctac atctg 35 8 33 DNA Artificial SequenceOligonucleotide 8 cuacuacuac uaggtaccct ctcgaaaagt taa 33 9 52 DNAArtificial Sequence Oligonucleotide 9 caucaucauc augaggaatt cagtgatggtgatggtgatg tgcaaaaaga gg 52 10 41 DNA Artificial SequenceOligonucleotide 10 actggaattc atgccaatcc atcaccatca ccatcacccg t 41 1141 DNA Artificial Sequence Oligonucleotide 11 acgtgtcgac catatggatgactaggtgaa acgggtgatg g 41 12 71 DNA Artificial Sequence Annealed primerproduct 12 actggaattc atgccaatcc atcaccatca ccatcacccg tttcacctagtcatccatat 60 ggtcgacacg t 71 13 48 DNA Artificial SequenceOligonucleotide 13 gactagttct agatcgcgag cggccgccca ttaactctcg ttggcagc48 14 16 DNA Artificial Sequence Oligonucleotide 14 tcgacccacg cgtccg 1615 12 DNA Artificial Sequence Oligonucleotide 15 cggacgcgtg gg 12 16 42DNA Artificial Sequence Oligonucleotide 16 auggagaucu cugaattcatgactgttgcg ctacatctgg ct 42 17 32 DNA Artificial SequenceOligonucleotide 17 attattcata tgactgttgc gctacatctg gc 32 18 25 DNAArtificial Sequence Oligonucleotide 18 tacgatctct ctccaggcca ttttc 25 1924 DNA Artificial Sequence Oligonucleotide 19 actcgagcag cccgggaacc tttg24 20 48 DNA Artificial Sequence Oligonucleotide 20 attacccgggaggatatcat atgttagcga tgacaatgga acataaag 48 21 36 DNA ArtificialSequence Oligonucleotide 21 atatgtcgac tcacagtggc cctccctata aatttg 3622 35 DNA Artificial Sequence Oligonucleotide 22 tattaggatc ccatgactgttgcgctacat ctggc 35 23 29 DNA Artificial Sequence Oligonucleotide 23gcaatccttg agctctaaga ccatcaggg 29 24 36 DNA Artificial SequenceOligonucleotide 24 ggacccactg tctttaccgc ggcctcctca agcacc 36

What is claimed is:
 1. A composition for use in reverse transcription ofa nucleic acid molecule, said composition comprising two or morepolypeptides having reverse transcriptase activity.
 2. The compositionof claim 1, wherein said polypeptides are obtained from differentsources.
 3. The composition of claim 1, wherein the transcription pausesite of each of said polypeptides is different from that of each of theother polypeptides in said composition.
 4. The composition of claim 1,wherein said polypeptides are reduced or substantially reduced in RNaseH activity.
 5. The composition of claim 4, wherein said polypeptides areselected from the group consisting of M-MLV H⁻ reverse transcriptase,RSV H⁻ reverse transcriptase, AMV H⁻ reverse transcriptase, RAV H⁻reverse transcriptase, MAV H⁻ reverse transcriptase and HIV H⁻ reversetranscriptase, and derivatives, variants, fragments or mutants thereof.6. The composition of claim 5, wherein said AMV H⁻ reverse transcriptaseis selected from the group consisting of AMV αH⁻/βH⁻ reversetranscriptase, AMV αH⁻/βH⁺ reverse transcriptase, AMV βH⁻/βH⁻ reversetranscriptase, AMV βH⁺/βH⁻ reverse transcriptase, AMV βp4/βp4 reversetranscriptase, and AMV αH⁻ reverse transcriptase, and derivatives,variants, fragments or mutants thereof.
 7. The composition of claim 5,wherein said RSV H⁻ reverse transcriptase is selected from the groupconsisting of RSV αH⁻/βH⁻ reverse transcriptase, RSV αH⁻/βH⁺ reversetranscriptase, RSV βH⁻/βH⁻ reverse transcriptase, RSV βH⁺/βH⁻ reversetranscriptase, RSV βp4/βp4 reverse transcriptase, and RSV αH⁻ reversetranscriptase, and derivatives, variants, fragments or mutants thereof.8. The composition of claim 1, wherein said polypeptides are selectedfrom the group consisting of Taq, Tne, Tma, Pfu, VENT™, DEEPVENT™ andTth DNA polymerases, and mutants, fragments, variants and derivativesthereof.
 9. The composition of claim 1, wherein said polypeptides arepresent in said composition at working concentrations.
 10. A method forreverse transcription of one or more nucleic acid molecules comprising(a) mixing one or more nucleic acid templates with two or morepolypeptides having reverse transcriptase activity; and (b) incubatingsaid mixture under conditions sufficient to make one or more firstnucleic acid molecules complementary to all or a portion of said one ormore templates.
 11. The method of claim 10, wherein said nucleic acidtemplate is a messenger RNA molecule or a population of mRNA molecules.12. The method of claim 10, said method further comprising incubatingsaid one or more first nucleic acid molecules under conditionssufficient to make one or more second nucleic acid moleculescomplementary to all or a portion of said one or more first nucleic acidmolecules.
 13. A cDNA molecule made according to the method of claim 10.14. A cDNA molecule made according to the method of claim
 12. 15. Amethod for amplifying one or more nucleic acid molecules, said methodcomprising (a) mixing one or more nucleic acid templates with two ormore polypeptides having reverse transcriptase activity and one or moreDNA polymerases; and (b) incubating said mixture under conditionssufficient to amplify one or more nucleic acid molecules complementaryto all or a portion of said one or more templates.
 16. A method foramplifying one or more nucleic acid molecules, said method comprising(a) mixing one or more nucleic acid templates with two or morepolypeptides having reverse transcriptase activity and DNA polymeraseactivity; and (b) incubating said mixture under conditions sufficient toamplify one or more nucleic acid molecules complementary to all or aportion of said one or more nucleic acid templates.
 17. A nucleic acidmolecule amplified according to the method of claim 15 or claim
 16. 18.A vector comprising the cDNA molecule of claim
 14. 19. The vector ofclaim 18, wherein said vector is an expression vector.
 20. A host cellcomprising the cDNA molecule of claim
 14. 21. A method for sequencingone or more nucleic acid molecules, said method comprising (a) mixingone or more nucleic acid molecules to be sequenced with one or moreprimers, two or more polypeptides having reverse transcriptase activity,one or more nucleotides and one or more terminating agents; (b)incubating said mixture under conditions sufficient to synthesize apopulation of molecules complementary to all or a portion of said one ormore molecules to be sequenced; and (c) separating said population todetermine the nucleotide sequence of all or a portion of said one ormore molecules to be sequenced.
 22. A kit for use in reversetranscription, amplification or sequencing of a nucleic acid molecule,said kit comprising two or more polypeptides having reversetranscriptase activity.
 23. The kit of claim 22, said kit furthercomprising one or more components selected from the group consisting ofone or more nucleotides, one or more DNA polymerases, a suitable buffer,one or more primers and one or more terminating agents.
 24. The kit ofclaim 23, wherein said terminating agent is a dideoxynucleotide.
 25. Thekit of claim 23, wherein two or more of the components of said kit arepresent as a mixture or are present as separate components.
 26. A methodof producing an ASLV reverse transcriptase, said method comprising (a)obtaining a host cell comprising one or more nucleic acid sequencesencoding one or more subunits of ASLV reverse transcriptase; and (b)culturing said host cell under conditions sufficient to produce saidASLV reverse transcriptase subunits.
 27. The method of claim 26, whereinsaid one or more nucleic acid sequences encoding one or more subunits ofASLV reverse transcriptase are contained in one or more vectors.
 28. Themethod of claim 26, wherein said ASLV reverse transcriptase subunits areselected from the group consisting of one or more a subunits, one ormore β subunits, and one or more βp4 subunits, of one or more ASLVreverse transcriptases, and derivatives, variants, fragments or mutantsthereof.
 29. The method of claim 26, wherein said subunits are one ormore α subunits.
 30. The method of claim 26, wherein said subunits areone or more β subunits.
 31. The method of claim 26, wherein saidsubunits are one or more βp4 subunits.
 32. The method of claim 26,wherein said subunits are one α subunit and one β subunit of one or moreASLV reverse transcriptases.
 33. The method of claim 26, wherein saidsubunits are co-expressed to form an ASLV reverse transcriptase andwherein said ASLV reverse transcriptase is isolated from said host cell.34. The method of claim 26, wherein said subunits of ASLV reversetranscriptase are isolated and then mixed to form an ASLV reversetranscriptase.
 35. The method of claim 30, wherein said p subunits forman ASLV reverse transcriptase comprising two β subunits.
 36. The methodof claim 32, wherein said α and β subunits form an ASLV reversetranscriptase comprising an α and a β subunit.
 37. The method of claim26, wherein one or more of said subunits have been modified to reduce orsubstantially reduce the RNase H activity in said subunits.
 38. Themethod of claim 26, wherein said subunits are encoded by one or morenucleotide sequences contained on the same or on different vectors. 39.The method of claim 26, wherein said ASLV reverse transcriptase is anRSV reverse transcriptase.
 40. The method of claim 26, wherein said ASLVreverse transcriptase is an AMV reverse transcriptase.
 41. An ASLVreverse transcriptase produced according to the method of claim
 26. 42.The ASLV reverse transcriptase of claim 41, wherein said ASLV reversetranscriptase is selected from the group consisting of an ASLV αβreverse transcriptase, an ASLV ββ reverse transcriptase, an ASLV βp4βp4reverse transcriptase, and an ASLV α reverse transcriptase.
 43. The ASLVreverse transcriptase of claim 41, wherein said ASLV reversetranscriptase is reduced or substantially reduced in RNase H activity.44. The ASLV reverse transcriptase of claim 41, wherein said ASLVreverse transcriptase is an RSV reverse transcriptase.
 45. The ASLVreverse transcriptase of claim 41, wherein said ASLV reversetranscriptase is an AMV reverse transcriptase.
 46. An isolated nucleicacid molecule comprising a nucleotide sequence encoding one or moresubunits of ASLV reverse transcriptase.
 47. The nucleic acid molecule ofclaim 46, wherein said molecule encodes one or more α subunits of ASLVreverse transcriptase, or a derivative, fragment or mutant thereof. 48.The nucleic acid molecule of claim 46, wherein said molecule encodes oneor more β subunits of ASLV reverse transcriptase, or a derivative,fragment or mutant thereof.
 49. The nucleic acid molecule of claim 46,wherein said molecule encodes one or more βp4 subunits of ASLV reversetranscriptase, or a derivative, fragment or mutant thereof.
 50. Thenucleic acid molecule of claim 46, wherein said molecule encodes α and βASLV reverse transcriptase subunits, or derivatives, variants, fragmentsor mutants thereof.
 51. A vector comprising the nucleic acid molecule ofclaim
 46. 52. A host cell comprising the nucleic acid molecule of claim46.
 53. The vector of claim 51, wherein said vector is plasmidpDABH−His.
 54. The host cell of claim 52, wherein said host cell is E.coli DH10B(pDABH−His).
 55. The isolated nucleic acid molecule of claim46, wherein said ASLV reverse transcriptase is an RSV reversetranscriptase.
 56. The isolated nucleic acid molecule of claim 46,wherein said ASLV reverse transcriptase is an AMV reverse transcriptase.57. A method for producing one or more cDNA molecules by reversetranscription of one or more nucleic acid templates comprising (a)mixing one or more nucleic acid templates with a ASLV RT comprising oneor more subunits; and (b) incubating said mixture under conditionssufficient to make one or more first nucleic acid moleculescomplementary to all or a portion of said one or more templates.
 58. Themethod of claim 57, wherein said subunits are one or more a subunits,one or more β subunits, one or more βp4 subunits, or a combinationthereof.
 59. The method of claim 57, wherein said ASLV reversetranscriptase is selected from the group consisting of an ASLV αβreverse transcriptase, an ASLV ββ reverse transcriptase, an ASLV βp4βp4reverse transcriptase, and an ASLV α reverse transcriptase.
 60. Themethod of claim 57, wherein said nucleic acid template is a mRNAmolecule or a population of mRNA molecules.
 61. The method of claim 57,wherein said method further comprises incubating said one or more firstnucleic acid molecules under conditions sufficient to make one or moresecond nucleic acid molecules complementary to all or a portion of saidone or more first nucleic acid molecules.
 62. The method of claim 61,wherein said first and said second nucleic acid molecules form a doublestranded DNA molecule.
 63. The method of claim 62, wherein said doublestranded DNA molecule is a full-length cDNA molecule.
 64. A cDNAmolecule made according to the method of claim
 57. 65. A cDNA moleculemade according to the method of claim
 61. 66. A vector comprising thecDNA molecule of claim
 65. 67. The vector of claim 66, wherein saidvector is an expression vector.
 68. A host cell comprising the cDNAmolecule of claim
 65. 69. The method of claim 61, wherein said ASLVreverse transcriptase is an RSV reverse transcriptase.
 70. The method ofclaim 61, wherein said ASLV reverse transcriptase is an AMV reversetranscriptase.
 71. A method for amplifying one or more nucleic acidmolecules comprising (a) mixing one or more nucleic acid templates withone or more ASLV RTs comprising one or more subunits and optionally withone or more DNA polymerases; and (b) incubating said mixture underconditions sufficient to amplify one or more nucleic acid moleculescomplementary to all or a portion of said one or more templates.
 72. Themethod of claim 71, wherein said subunits are one or more a subunits,one or more β subunits, one or more βp4 subunits, or a combinationthereof.
 73. The method of claim 71, wherein said ASLV reversetranscriptase is selected from the group consisting of an ASLV αβreverse transcriptase, an ASLV ββ reverse transcriptase, an ASLV βp4βp4reverse transcriptase, and an ASLV α reverse transcriptase.
 74. Themethod of claim 71, wherein said ASLV reverse transcriptase is an RSVreverse transcriptase.
 75. The method of claim 71, wherein said ASLVreverse transcriptase is an AMV reverse transcriptase.
 76. A method forsequencing one or more nucleic acid molecules comprising (a) mixing oneor more nucleic acid molecules to be sequenced with one or more primers,an ASLV RT comprising one or more subunits, one or more nucleotides andone or more terminating agents; (b) incubating said mixture underconditions sufficient to synthesize a population of nucleic acidmolecules complementary to all or a portion of said one or more nucleicacid molecules to be sequenced; and (c) separating said population ofnucleic acid molecules to determine the nucleotide sequence of all or aportion of said one or more nucleic acid molecules- to be sequenced. 77.The method of claim 76, wherein said subunits are one or more asubunits, one or more β subunits, one or more βp4 subunits, or acombination thereof.
 78. The method of claim 76, wherein said ASLVreverse transcriptase is selected from the group consisting of an ASLVαβ reverse transcriptase, an ASLV ββ reverse transcriptase, an ASLVβp4βp4 reverse transcriptase, and an ASLV α reverse transcriptase. 79.The method of claim 76, wherein said ASLV reverse transcriptase is anRSV reverse transcriptase.
 80. The method of claim 76, wherein said ASLVreverse transcriptase is an AMV reverse transcriptase.
 81. A kitcomprising one or more ASLV RT subunits, or one or more derivatives,variants, fragments or mutants thereof.
 82. The kit of claim 81, whereinsaid ASLV RT subunits are one or more ASLV RT α subunits, one or moreASLV RT β subunits, one or more ASLV RT βp4 subunits, or a combinationthereof.
 83. The kit of claim 81, wherein said ASLV reversetranscriptase is an RSV reverse transcriptase.
 84. The kit of claim 81,wherein said ASLV reverse transcriptase is an AMV reverse transcriptase.85. A kit comprising an ASLV RT, wherein said ASLV RT is selected fromthe group consisting of an ASLV αβ RT, an ASLV ββ RT, an ASLV βp4βp4 RT,and an ASLV α RT, or a derivative, fragment or mutant thereof.
 86. Thekit of claim 85, wherein said ASLV RT is comprises an α and a β subunit.87. The kit of claim 85, wherein said ASLV reverse transcriptase is anRSV reverse transcriptase.
 88. The kit of claim 85, wherein said ASLVreverse transcriptase is an AMV reverse transcriptase.
 89. A method forproducing one or more cDNA molecules by reverse transcription of one ormore nucleic acid templates comprising (a) mixing one or more nucleicacid templates with one or more polypeptides having reversetranscriptase activity; and (b) incubating said mixture at a temperatureof about 50° C. or greater and under conditions sufficient to make oneor more first nucleic acid molecules complementary to all or a portionof said one or more templates.
 90. The method of claim 89, wherein saidtemperature is 60° C. or greater.
 91. The method of claim 89, whereinsaid temperature ranges from about 50° C. to about 70° C.
 92. The methodof claim 89, wherein said temperature ranges from about 55° C. to about65° C.
 93. The method of claim 89, wherein said nucleic acid template isan RNA or DNA molecule.
 94. The method of claim 93, wherein said RNAmolecule is an mRNA molecule or a polyA+ RNA molecule.
 95. The method ofclaim 89, wherein said nucleic acid template is a population of mRNAmolecules.
 96. The method of claim 94, wherein said first nucleic acidmolecule is a full length cDNA molecule.
 97. The method of claim 89,further comprising incubating said one or more first nucleic acidmolecules under conditions sufficient to make one or more second nucleicacid molecules complementary to all or portion of said one or more firstnucleic acid molecules.
 98. The method of claim 97, wherein said firstand said second nucleic acid molecules are DNA molecules.
 99. The methodof claim 98, wherein said first and said second DNA molecules form adouble stranded DNA molecule.
 100. The method of claim 99, wherein saiddouble stranded DNA molecule is a full length cDNA molecule.
 101. Themethod of claim 89, wherein said one or more polypeptides having reversetranscriptase activity are reduced or substantially reduced in RNase Hactivity.
 102. The method of claim 89, wherein said one or morepolypeptides having reverse transcriptase activity is one or more ASLVreverse transcriptases comprising one or more subunits.
 103. The methodof claim 102, wherein said one or more ASLV reverse transcriptases isone or more RSV reverse transcriptases.
 104. The method of claim 102,wherein said one or more ASLV reverse transcriptases is one or more AMVreverse transcriptases.
 105. The method of claim 102, wherein said ASLVRT subunits are one or more ASLV RT α subunits, one or more ASLV RT βsubunits, one or more ASLV RT βp4 subunits, or a combination thereof.106. The method of claim 102, wherein said ASLVRT is selected from thegroup consisting of an ASLV αβ RT, an ASLV ββ RT, an ASLV βp4βp4 RT, andan ASLV α RT, and derivatives, variants, fragments or mutants thereof.107. The method of claim 102, wherein said ASLV RT is ASLV αβ RT, or aderivative, variant, fragment or mutant thereof.
 108. The method ofclaim 102, wherein said one or more subunits are reduced orsubstantially reduced in RNase H activity.
 109. A nucleic acid moleculeproduced according to the method of claim
 89. 110. A nucleic acidmolecule produced according to the method of claim
 97. 111. The nucleicacid molecule of claim 110, wherein said nucleic acid molecule is afull-length cDNA molecule.
 112. A vector comprising the nucleic acidmolecule of claim
 110. 113. The vector of claim 112, wherein said vectoris an expression vector.
 114. A host cell comprising the nucleic acidmolecule of claim
 109. 115. A host cell comprising the nucleic acidmolecule of claim 110.